Expansion and genetic modification of human natural killer cells for adoptive immunotherapy of cancer

A 
century 
after 
the 
initial
 proposition 
that 
the 
immune
 system 
has 
the
 capacity 
to
 fight
 against 
tumors, 
evading 
destruction
 by 
immune
 cells
 is 
now
 well 
recognized
 as
 a
 hallmark
 of
 cancer.
 Recent
 decades
 have
 witnessed
 extraordinary
 improvements 
in
 the 
use
 of
 immunotherapy 
against 
malignancies 
and
 adoptive
 transfer
 of
 Natural
 Killer
 (NK)
 cells
 stands
 among
 promising
 tools
 in
 the
 fight
 against
 cancer.
 Clinical
 studies
 have
 demonstrated
 the
 anti‐tumor
 responses
 generated 
by
 NK 
cells 
both 
in
 the
 autologous 
and
 allogeneic
 settings
 in
 various
 cancers.
Direct 
adoptive 
transfer,
 ex
 vivo
 activation 
and/o r
expansion, 
as 
well
 as
 genetic
 modification
 of
 NK
 cells
 aspire
 novel
 improvements
 to
 current
 immunotherapy
 strategies.
 As
 such
 interventions
 develop,
 the
 quest
 for
 better
 preparation
 of 
NK 
cell
 based 
therapies 
continues. This 
thesis, 
primarily
 investigates 
the
 feasibility
 and 
potential
 of
 ex
 vivo
 expanded
 NK
 cells
 for
 cancer 
immunotherapy. 
Our 
results
p roduced
 a 
system 
that
 has 
the
 capacity 
to
 expand
 polyclonal 
and 
highly 
cytotoxic
 NK 
cells showing 
selective
 anti‐ tumor
 activity.
 Protocols
 for
 expansion
 of
 these
 cells
 from
 healthy
 donors
 and
 patients
 with
 Multiple
 Myeloma
 (MM)
 using
 current
 Good
 Manufacturing
 Practice
 (cGMP)‐compliant
 methods
 have
 been
 optimized
 in
 conventional
 cell
 culture 
systems 
as
 well
 as
automated
 bioreactors.
The
 elevated
 cytotoxic
activity
 of 
expanded 
NK
 cells 
against
 autologous
 tumor 
cells,
 along
 with
 detailed
 analysis
 of
 phenotypic
 changes
 during
 the
 expansion
 process
 has
 subsequently
 shifted
 attention 
to
 the
 interaction 
between 
NK 
and
 tumor 
cells. Both 
as
 a 
basic 
method 
to
 identify
 these
 interactions,
 and 
as 
part 
of
 further
 plans
 to
 use
 genetically
 retargeted
 NK
 cells
 in
 cancer
 immunotherapy,
 we
 have
 investigated
 methods
 for 
efficient
 lentiviral
 genetic
 modification 
of
 NK 
cells.
This
 study 
has 
resulted 
in
 an
 optimized 
stimulation 
and
 genetic
 modification 
process
 for
 NK
 cells
 that
 greatly
 enhances
 viral
 gene
 delivery.
 Along
 with
 NK
 cell
 stimulating
 cytokines,
 an
 inhibitor
 of
 innate
 immune
 receptor
 signaling
 that
 blocks
 the
 intracellular
 detection
 of
 viral
 RNA
 introduced
 by
 the
 vector
 was
 successfully
 utilized 
to 
enhance 
gene
 transfer 
efficiency,
 also
 constituting 
a
 proof‐ of‐concept 
for
 various
 other
 gene
 therapy
 approaches. Taken
 together,
 the
 work
 presented
 in
 this
 thesis
 aims
 to
 bring
 us
 closer
 to
 optimal
 ex 
vivo
 manipulation 
of
 NK 
cells
 for
 immunotherapy.
 Clinical 
trials 
with
 the 
long‐term
 expanded 
NK 
cells
 as
 well
 as 
further 
preclinical 
development
 of 
NK
 cell
 genetic 
modification 
processes 
are
 warranted

Deletion of chromosomal region 8p21 confers resistance to Bortezomib and is associated with upregulated Decoy trail receptor expression in patients with multiple myeloma

Loss of the chromosomal region 8p21 negatively effects survival in patients with multiple myeloma (MM) that undergo autologous stem cell transplantation (ASCT). In this study, we aimed to identify the immunological and molecular consequences of del(8)(p21) with regards to treatment response and bortezomib resistance. In patients receiving bortezomib as a single first line agent without any high-dose therapy, we have observed that patients with del(8)(p21) responded poorly to bortezomib with 50% showing no response while patients without the deletion had a response rate of 90%. In vitro analysis revealed a higher resistance to bortezomib possibly due to an altered gene expression profile caused by del(8)(p21) including genes such as TRAIL-R4, CCDC25, RHOBTB2, PTK2B, SCARA3, MYC, BCL2 and TP53. Furthermore, while bortezomib sensitized MM cells without del(8)(p21) to TRAIL/APO2L mediated apoptosis, in cells with del(8)(p21) bortezomib failed to upregulate the pro-apoptotic death receptors TRAIL-R1 and TRAIL-R2 which are located on the 8p21 region. Also expressing higher levels of the decoy death receptor TRAIL-R4, these cells were largely resistant to TRAIL/APO2L mediated apoptosis. Corroborating the clinical outcome of the patients, our data provides a potential explanation regarding the poor response of MM patients with del(8)(p21) to bortezomib treatment. Furthermore, our clinical analysis suggests that including immunomodulatory agents such as Lenalidomide in the treatment regimen may help to overcome this negative effect, providing an alternative consideration in treatment planning of MM patients with del(8)(p21)

Circulating LL37 targets plasma extracellular vesicles to immune cells and intensifies Behçet's disease severity

Behçet's disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. © 2017 The Author(s)

Membrane-Bound IL-21 Promotes Sustained Ex Vivo Proliferation of Human Natural Killer Cells

NK cells have therapeutic potential for a wide variety of human malignancies. However, because NK cells expand poorly in vitro, have limited life spans in vivo, and represent a small fraction of peripheral white blood cells, obtaining sufficient cell numbers is the major obstacle for NK-cell immunotherapy. Genetically-engineered artificial antigen-presenting cells (aAPCs) expressing membrane-bound IL-15 (mbIL15) have been used to propagate clinical-grade NK cells for human trials of adoptive immunotherapy, but ex vivo proliferation has been limited by telomere shortening. We developed K562-based aAPCs with membrane-bound IL-21 (mbIL21) and assessed their ability to support human NK-cell proliferation. In contrast to mbIL15, mbIL21-expressing aAPCs promoted log-phase NK cell expansion without evidence of senescence for up to 6 weeks of culture. By day 21, parallel expansion of NK cells from 22 donors demonstrated a mean 47,967-fold expansion (median 31,747) when co-cultured with aAPCs expressing mbIL21 compared to 825-fold expansion (median 325) with mbIL15. Despite the significant increase in proliferation, mbIL21-expanded NK cells also showed a significant increase in telomere length compared to freshly obtained NK cells, suggesting a possible mechanism for their sustained proliferation. NK cells expanded with mbIL21 were similar in phenotype and cytotoxicity to those expanded with mbIL15, with retained donor KIR repertoires and high expression of NCRs, CD16, and NKG2D, but had superior cytokine secretion. The mbIL21-expanded NK cells showed increased transcription of the activating receptor CD160, but otherwise had remarkably similar mRNA expression profiles of the 96 genes assessed. mbIL21-expanded NK cells had significant cytotoxicity against all tumor cell lines tested, retained responsiveness to inhibitory KIR ligands, and demonstrated enhanced killing via antibody-dependent cell cytotoxicity. Thus, aAPCs expressing mbIL21 promote improved proliferation of human NK cells with longer telomeres and less senescence, supporting their clinical use in propagating NK cells for adoptive immunotherapy

Revving up natural killer cells and cytokine-induced killer cells against hematological malignancies

Natural killer (NK) cells belong to innate immunity and exhibit cytolytic activity against infectious pathogens and tumor cells. NK-cell function is finely tuned by receptors that transduce inhibitory or activating signals, such as killer immunoglobulin-like receptors, NK Group 2 member D (NKG2D), NKG2A/CD94, NKp46, and others, and recognize both foreign and self-antigens expressed by NK-susceptible targets. Recent insights into NK-cell developmental intermediates have translated into a more accurate definition of culture conditions for the in vitro generation and propagation of human NK cells. In this respect, interleukin (IL)-15 and IL-21 are instrumental in driving NK-cell differentiation and maturation, and hold great promise for the design of optimal NK-cell culture protocols. Cytokine-induced killer (CIK) cells possess phenotypic and functional hallmarks of both T cells and NK cells. Similar to T cells, they express CD3 and are expandable in culture, while not requiring functional priming for in vivo activity, like NK cells. CIK cells may offer some advantages over other cell therapy products, including ease of in vitro propagation and no need for exogenous administration of IL-2 for in vivo priming. NK cells and CIK cells can be expanded using a variety of clinical-grade approaches, before their infusion into patients with cancer. Herein, we discuss GMP-compliant strategies to isolate and expand human NK and CIK cells for immunotherapy purposes, focusing on clinical trials of adoptive transfer to patients with hematological malignancies

Poly(I:C) Enhances the Susceptibility of Leukemic Cells to NK Cell Cytotoxicity and Phagocytosis by DC

α Active specific immunotherapy aims at stimulating the host's immune system to recognize and eradicate malignant cells. The concomitant activation of dendritic cells (DC) and natural killer (NK) cells is an attractive modality for immune-based therapies. Inducing immunogenic cell death to facilitate tumor cell recognition and phagocytosis by neighbouring immune cells is of utmost importance for guiding the outcome of the immune response. We previously reported that acute myeloid leukemic (AML) cells in response to electroporation with the synthetic dsRNA analogue poly(I:C) exert improved immunogenicity, demonstrated by enhanced DC-activating and NK cell interferon-γ-inducing capacities. To further invigorate the potential of these immunogenic tumor cells, we explored their effect on the phagocytic and cytotoxic capacity of DC and NK cells, respectively. Using single-cell analysis, we assessed these functionalities in two- and three-party cocultures. Following poly(I:C) electroporation AML cells become highly susceptible to NK cell-mediated killing and phagocytosis by DC. Moreover, the enhanced killing and the improved uptake are strongly correlated. Interestingly, tumor cell killing, but not phagocytosis, is further enhanced in three-party cocultures provided that these tumor cells were upfront electroporated with poly(I:C). Altogether, poly(I:C)-electroporated AML cells potently activate DC and NK cell functions and stimulate NK-DC cross-talk in terms of tumor cell killing. These data strongly support the use of poly(I:C) as a cancer vaccine component, providing a way to overcome immune evasion by leukemic cells

Processing unit resources in a distributed computing systems

The described program is designed for collecting, storing and processing data of distributed file system

The factors that cause the misjudgement in questioned document examination: Extraneous marks and deteriorating factors

Artificial defects on the documents can be classified into two categories: extraneous marks and deteriorating factors. Identifying and categorizing artificial defects of the examined document and determining some critical unforeseen details are the aims of this study. Real case samples were collected from our archive and examined. It was found that 41 out of total 100 cases include either extraneous marks or deteriorating factors.Artificial defects on the documents can be classified into two categories: extraneous marks and deteriorating factors. Identifying and categorizing artificial defects of the examined document and determining some critical unforeseen details are the aims of this study. Real case samples were collected from our archive and examined. It was found that 41 out of total 100 cases include either extraneous marks or deteriorating factors