Note: Descriptions are shown in the official language in which they were submitted.
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CIML NK CELLS AND METHODS THEREFOR
Field of the Invention
[0001] The present disclosure relates to compositions, methods, and devices to
generate
and/or cultivate activated immune competent cells, especially as it relates
memory like NK
cells that are produced from cord blood (CB) or peripheral blood (PB).
Back2round of the Invention
[0002] The background description includes information that may be useful in
understanding
the present disclosure. It is not an admission that any of the information
provided herein is
prior art or relevant to the presently claimed invention, or that any
publication specifically or
implicitly referenced is prior art.
[0003] All publications and patent applications herein are incorporated by
reference to the
same extent as if each individual publication or patent application were
specifically and
individually indicated to be incorporated by reference. Where a definition or
use of a term in
an incorporated reference is inconsistent or contrary to the definition of
that term provided
herein, the definition of that term provided herein applies and the definition
of that term in the
reference does not apply.
[0004] Natural killer (NK) cells constitute a group of innate immune cells,
which are often
characterized as cytotoxic lymphocytes that exhibit antibody dependent
cellular toxicity via
target-directed release of granulysin and perforin. Most NK cells have a
specific cell surface
marker profile (e.g., CD3 , CD56+, CD16+, CD57+, CD8+) in addition to a
collection of
various activating and inhibitory receptors. While more recently NK cells have
become a
significant component of certain cancer treatments, generation of significant
quantities of NK
cells (and especially autologous NK cells) has been a significant obstacle as
the fraction of
NK cells in whole blood is relatively low.
[0005] To obtain therapeutically meaningful quantities of NK and NK-like
cells, NK cells
can be generated from various precursor cells. For example, various stem cell
factors (SCF),
FLT3 ligand, interleukin (IL)-2, IL-7 and IL-15 have been reported in various
in vitro
approaches to induce and expand cord blood-derived cytokine-induced killer
(CIK) cells
(Anticancer Research 30: 3493-3500 (2010)). Similarly, CD34+ hematopoietic
cells can be
exposed to IL-12 and other agents as is reported in US 2018/0044636. In still
other
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approaches, human hemangioblasts were sequentially exposed to two different
cytokine
cocktails as described in W02011/068896, and different cytokine cocktails were
used with
post-embryonic hematopoietic stem cells as taught in W02012/128622. While at
least some
of these methods provide a significant n-fold expansion of NK cells, methods
and reagents
for such expansion are both time and resource demanding. Still further, it
should be noted
that many of the known methods also require NK cell culture on a feeder cell
layer, which is
often problematic from a technical and a regulatory perspective.
[0006] In more simplified methods, acute myeloid leukemia (AML) cells can be
exposed to
TpoR agonists to so induce the AML cells to form NK cells. However, such
approach is
likely not viable as a source for therapeutic cell preparations. Alternative
methods have also
relied on culturing peripheral blood cells in the presence of various
interleukins, stem cell
factors, and FLT3 ligands as is disclosed in WO 2011/103882. In yet another
method, US
2013/0295671 teaches methods of stimulating already existing NK cells with
anti-CD16 and
anti-CD3 antibodies along with cytokines. While procedurally simpler, such
methods still
require elaborate manipulation of the cells and add significant costs due to
the specific
reagent required.
[0007] In still further known methods, US 10,125,351 describes use of cord
blood or
peripheral blood as a source of cells that are subject to density gradient
separation to isolate
nucleated cells that are then cultivated with a medium that contains
interferon, interleukin, a
CD3 antibody and human albumin. Most advantageously, such method is amenable
to
perfusion culture in a bioreactor and as such significantly reduces
operational difficulties.
Unfortunately, however, the yield of NK cells is relatively low.
[0008] Regardless of the specific manner of production, cultivated NK cells
will typically not
exhibit memory like character, which is particularly desirable for cancer
immune therapy. In
at least some attempts to produce memory like NK cells, cultivated NK cells
were exposed to
IL-12, IL-15, and IL-18 and so exposed NK cells exhibited a memory like
phenotype and
correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of
CD57
and MR (see Blood (2012) Vol.120, No.24; 4751-4760). Similarly, memory like NK
cells
were prepared by pre-activating NK cells using various stimulatory cytokines
followed by
contacting the pre-activated cells with PM21 particles, EX21 exosomes, or FC21
feeder cells
as is described in WO 2018/089476. In yet another approach to generate memory
like NK
cells, freshly isolated NK cells were exposed to an IL-18/IL-12-TxM fusion
protein complex
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as is described in WO 2018/165208. While such methods typically produced at
least some
NK cells with memory-like character, the cytotoxicity of such activated NK
cells against
selected target cells was still less than optimal, possibly due to lack or low
expression of
specific activating receptors and/or expression of specific inhibitory
receptors.
[0009] Thus, even though various methods of generating memory like NK cells
are known in
the art, all or almost all of them suffer from various disadvantages.
Consequently, there is a
need to provide improved systems and methods that produce memory like NK
cells, and
especially autologous memory like NK cells in significant quantities.
Moreover, improved
systems and methods will also allow for automation of cell culture and NK cell
activation and
will have substantially reduced reagent requirements to render such methods
clinically and
commercially viable.
Summary of The Invention
[0010] The inventors have discovered compositions, methods, and devices that
enable
generation and expansion of memory like NK cells in a conceptually simple and
efficient
manner. Advantageously, memory like NK cells can be generated in a 2-step
process in
which NK cells are expanded to a desired quantity and in which the expanded NK
cells are
then induced with a mixture of cytokines to so form the cytokine induced
memory like
(CIML) NK cells. Expansion of the NK cells is preferably performed in an
enrichment
process that uses N-803 and an anti-CD16 agonist antibody and optionally an
anti-CD3
antibody. Activation to obtain the memory like character is then performed
with a
combination of stimulatory cytokines, most preferably with IL-12/IL-15/IL-18
or an IL-
18/IL-12-TxM fusion protein complex.
[0011] Unexpectedly, and besides upregulation of activation markers and IFN-y
secretion, so
activated expanded memory like NK cells had an increased expression of CD25
and the NK
activation receptor DNAM-1 and a downregulated expression of the inhibitory
receptor,
TIGIT, which presumably contributed or even caused the observed heightened
toxicity
against of the CIML NK cells. Most notably, the CIML NK cells presented herein
even
exhibited significant cytotoxicity against the otherwise NK resistant tumor
cell line MS-1 at a
relatively low effector to target ratio.
[0012] In one aspect of the inventive subject matter, the inventors
contemplate a method of
producing CIML NK cells with enhanced cytotoxicity that includes one step of
isolating from
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a biological fluid a mixture of mononuclear cells, and another step of
contacting the mixture
of the mononuclear cells with an anti-CD16 antibody and N-803 to expand NK
cells. In a
further step, the expanded NK cells are contacted with a stimulatory cytokine
composition
(typically including an IL-18/IL-12-TxM fusion protein complex, a mixture of
IL-12, N-803,
and IL-18, or a mixture of IL-12, IL-15, and IL-18) to thereby generate the
CIML NK cells
with enhanced cytotoxicity. Where desired, contemplated methods may further
comprise a
step of contacting the CIML NK cells with N-803 after re-stimulating the CIML
NK cells.
[0013] Preferably, but not necessarily, the biological fluid is whole blood or
cord blood, and
the mixture of mononuclear cells is not further processed to enrich NK cells.
Most typically,
the mixture of the mononuclear cells contains about 100-500 x 106 cells,
and/or the step of
contacting the mixture is performed in a volume of between about 100-300 ml or
at a cell
density of about 1 x 106 cells/ml. In further embodiments, the anti-CD16
antibody in the step
of contacting the mixture may be present at a concentration of between 0.05-
0.5 mcg/ml,
and/or the N-803 in the step of contacting the mixture may be present at a
concentration of
between 0.1-1.0 nM. Optionally, the step of contacting the mixture may further
include a step
of contacting the mixture of the mononuclear cells with an anti-CD3 antibody
(e.g., at an
anti-CD3 antibody concentration of between 0.1-1.0 ng/ml).
[0014] While in some aspects the stimulatory cytokine composition includes the
IL-18/IL-12-
TxM fusion protein complex, in other aspects the stimulatory cytokine
composition includes
the mixture of IL-12, N-803, and IL-18, and in further aspects the stimulatory
cytokine
composition includes the mixture of IL-12, IL-15, and IL-18. Most typically,
the NK cells are
expanded to a total cell number of about 0.5- 5.0 x 109 cells, and/or the step
of contacting the
expanded NK cells with the stimulatory cytokine composition is performed in
the same
container as the step of expanding the NK cells.
[0015] Therefore, and viewed from a different perspective, the inventors also
contemplate a
method of activating NK cells to form CIML NK cells with enhanced
cytotoxicity. Such
methods will include a step of providing expanded NK cells (typically expanded
from
mononuclear cells of whole blood or cord blood) and a further step of
contacting the
expanded NK cells with a stimulatory cytokine composition that may include an
IL-18/IL-12-
TxM fusion protein complex, a mixture of IL-12, N-803, and IL-18, or a mixture
of IL-12,
IL-15, and IL-18 to so generate the CIML NK cells with enhanced cytotoxicity.
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[0016] As noted before, it is contemplated that the NK cells are expanded from
whole blood
or from cord blood. Thus, the NK cells may be autologous relative to an
individual receiving
a transfusion comprising the CIML NK cells. Preferably, the stimulatory
cytokine
composition includes the IL-18/IL-12-TxM fusion protein complex. However, in
further
embodiments the stimulatory cytokine composition may also include a mixture of
IL-12, N-
803, and IL-18 or a mixture of IL-12, IL-15, and IL-18. Typically, the
expanded NK cells
have a total cell number of about 0.5- 5.0 x 109 cells.
[0017] It is further contemplated that the CIML NK cells with enhanced
cytotoxicity will
have cytotoxicity against MS-1 cells, that the CIML NK cells with enhanced
cytotoxicity
have a decreased expression of CD16 as compared to expanded NK cells that are
contacted
with N-803 alone, that the CIML NK cells with enhanced cytotoxicity have a
decreased
expression of TIGIT as compared to expanded NK cells that are contacted with N-
803 alone,
and/or that the CIML NK cells with enhanced cytotoxicity have an increased
expression of
CD25 and/or DNAM1 as compared to expanded NK cells that are contacted with N-
803
alone.
[0018] Therefore, the inventors also contemplate a CIML NK cell with enhanced
cytotoxicity
that exhibits cytotoxicity against MS-1 cells of at least 50% killing at an
effector to target cell
ratio of equal or less than 5. In further aspects, the CIML NK cell has a
decreased expression
of CD16 as compared to expanded NK cells that are contacted with N-803 alone,
has a
decreased expression of TIGIT as compared to expanded NK cells that are
contacted with N-
803 alone, and/or has an increased expression of CD25 and/or DNAM1 as compared
to
expanded NK cells that are contacted with N-803 alone.
[0019] While not limiting the inventive subject matter, the CIML NK cell is
preferably an
autologous cell relative to an individual receiving a transfusion comprising
the CIML NK
cell. In other embodiments, the CIML NK cell may also be a recombinant NK
cell. For
example, such recombinant cells may express CD16 or a variant thereof, IL-2 or
a variant
thereof, and/or IL-15 or a variant thereof from a recombinant nucleic acid.
[0020] In still further contemplated aspects, the inventors also contemplate a
pharmaceutical
composition comprising a pharmaceutically acceptable carrier in combination
with the CIML
NK cells as presented herein. Consequently, use of the CIML NK cell as
presented herein in
medicine, and particularly in the treatment of cancer is contemplated.
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[0021] Therefore, the inventors also contemplate a method of treating an
individual with a
CIML NK cell in need thereof that includes a step of administering to the
individual a
therapeutically effective amount of the CIML NK cell as presented herein.
Preferably, the
CIML NK cell is an autologous cell of the individual, and/or the CIML NK cell
is a
peripheral blood or cord blood derived NK cell.
[0022] Various objects, features, aspects, and advantages will become more
apparent from
the following detailed description of preferred embodiments, along with the
accompanying
drawing in which like numerals represent like components.
Brief Description of The Drawin2
[0023] Fig.1 depicts an exemplary schematic of an IL-18/IL-12-TxM fusion
protein complex.
[0024] Fig.2 depicts exemplary results from peripheral blood NK cell expansion
using
autologous PBMCs and selected combinations of specific antibodies.
[0025] Fig.3 depicts exemplary results from a cytotoxicity assay of cord blood
derived CIML
NK cells against MS-1 target cells.
[0026] Fig.4 depicts exemplary results for expression of selected phenotype
markers on cord
blood derived CIML NK cells.
[0027] Fig.5 depicts exemplary results from a cytotoxicity assay of peripheral
blood derived
CIML NK cells against MS-1 target cells.
[0028] Fig.6 depicts exemplary results for expression of selected phenotype
markers on
peripheral blood derived CIML NK cells.
[0029] Fig.7 depicts an exemplary activation cluster phenotype of cord blood
derived CIML
NK cells after IL-18/12 TxM exposure.
[0030] Fig.8 depicts an exemplary CD25 expression on cord blood derived CIML
NK cells
after IL-18/12 TxM exposure.
[0031] Fig.9 depicts an exemplary activation cluster phenotype of cord blood
derived CIML
NK cells upon restimulation.
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[0032] Fig.10A-10C depict exemplary results for cell killing activity of cord
blood derived
CIML NK cells after 24 hours (10A), 48 hours (10B), and 72 hours (10C) IL-
18/12 TxM
exposure.
[0033] Fig.11 depicts exemplary results for cell killing activity of cord
blood derived CIML
NK cells cultivated in a single-box culture environment and activation
clustering from such
cells.
[0034] Fig.12 depicts exemplary results for NK marker expression on peripheral
blood
derived CIML NK cells after IL-18/12 TxM exposure.
[0035] Fig.13 depicts exemplary results from a cytotoxicity assay of
peripheral blood derived
CIML NK cells after IL-18/12 TxM exposure against K562 target cells.
[0036] Fig.14 depicts exemplary results for IFN-y staining of peripheral blood
derived CIML
NK cells after re-stimulation.
[0037] Fig.15 depicts exemplary results from a cytotoxicity assay of
peripheral blood derived
CIML NK cells after exposure to N-803.
[0038] Fig.16 depicts exemplary results for IFN-y staining of cord blood
derived CIML NK
cells after re-stimulation and exemplary results from a cytotoxicity assay
after exposure to N-
803.
Detailed Description
[0039] Immune therapies in the treatment of cancer increasingly make use of
various cell-
based components, and more recently NK cells have become a promising modality.
While
some NK cells are now available at relatively high quantities, production of
therapeutically
meaningful quantities of autologous NK cells and/or memory like NK cells have
remained
problematic at best. Unfortunately, many of the current methods require use of
feeder layers
or differentiation of isolated CD34+ hematopoietic stem cells (HSCs), which is
both time and
resource intensive. Moreover, due to the various manipulation steps needed,
such methods
typically require human interaction and are prone to contamination. In
addition, conversion of
NK cells to a memory like phenotype may reduce cytotoxicity in at least some
protocols or
may not deliver sufficient amounts of such cells.
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[0040] The inventors have now discovered various systems, compositions, and
methods to
generate therapeutically meaningful quantities (e.g., at least 0.5 x 109 NK
cells) of NK cells
that can be readily converted to memory like NK cells in a simple and
effective manner that
can even be fully automated once the mononuclear cells are obtained from a
biological fluid
(e.g., whole blood, cord blood). Advantageously, such NK cells can be
autologous NK cells
and can be induced to a memory like phenotype to yield cytokine induced memory
like
(CIML) NK cells with enhanced cytotoxicity. Notably, and as is described in
more detail
below, the so generated CIML NK cells will have superior cytotoxicity as
compared to other
(CIML) NK cells and even have significant killing capacity against target
cells that are
otherwise resistant or even inert to NK cell cytotoxicity such as MS-1 cells
(Merkel cell
carcinoma cells).
[0041] While not wishing to be bound by any theory or hypothesis, the
inventors contemplate
that the enhanced cytotoxicity may be due to the source of (naïve) NK cells,
prior expansion
conditions, and possibly to the uninterrupted (e.g., change in media, culture
conditions, etc.)
nature of expansion and cytokine induction, which may result in the over-
expression of
activating factors and the under-expression of inhibitory receptors. Among
other notable
features of the CIML NK cells presented herein, the CIML NK cells will
typically exhibit
cytotoxicity against MS-1 cells of at least 50% killing at an effector to
target cell ratio of
equal or less than 5, will have a decreased expression of TIGIT (inhibitory
receptor) as
compared to expanded NK cells that are contacted with N-803 alone, and an
increased
expression of CD25 and/or DNAM1 (activating co-receptor) as compared to
expanded NK
cells that are contacted with N-803 alone. Thus, the term "NK cells with
enhanced
cytotoxicity" refers to NK cells that exhibit cytotoxicity against MS-1 cells
of at least 50%
killing at an effector to target cell ratio of equal or less than 5, a
decreased expression of
TIGIT (inhibitory receptor) as compared to expanded NK cells that are
contacted with N-803
alone, and/or an increased expression of CD25 and/or DNAM1 (activating co-
receptor) as
compared to expanded NK cells that are contacted with N-803 alone. Moreover,
contemplated CIML NK cells will typically also exhibit a decreased expression
of CD16.
Most typically, the CIML NK cells will exhibit all three of the above
parameters (i.e.,
cytotoxicity against otherwise NK resistant cells, increased expression of
activating receptors,
decreased expression of inhibitory receptors).
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[0042] In one exemplary process contemplated herein, NK cells are in a first
step expanded
from a fraction of a biological fluid containing mononuclear cells, preferably
to a total cell
number of about 0.5- 5.0 x 109 cells. Notably, such expansion can be performed
in a single
reactor in a relatively small volume to a moderate cell density (e.g., 100-300
ml or at a cell
density of about 0.5- 5.0 x 106 cells/m1) without the need for feeder cells or
other
manipulations that would require change of a culture vessel. Once a desired NK
cell quantity
is achieved, the so expanded NK cells are then in a second step contacted with
a stimulatory
cytokine composition to activate the NK cells to a memory like phenotype.
Preferably, but
not necessarily, the stimulatory cytokine composition will include an IL-18/IL-
12-TxM
fusion protein complex as is exemplarily depicted in Fig.!. However, the
stimulatory
cytokine composition may also include a mixture of IL-12, N-803, and IL-18, or
a mixture of
IL-12, IL-15, and IL-18. Cytokine stimulation will typically be performed for
a period of
between 4-24 hours, and the so generated CIML NK cells may be rested or re-
stimulated
(preferably in the presence of N-803) prior to transfusion.
[0043] For example, whole blood or cord blood can employed as a starting
material that is
processed to obtain mononuclear cells. Most typically, processing can be done
using
conventional density gradient centrifugation (e.g., using Ficoll-Paque PlusTM
(a hydrophilic
soluble polysaccharide, density 1.077 g/mL), commercially available from GE
Lifesciences).
Once the mononuclear cells are separated from the centrifuge tube, the cells
are washed and
re-suspended in an activation medium (e.g., NK MACS supplemented with 10%
human AB
serum). The activation medium can further comprise N-803 at a concentration of
about 0.4
nM, and an anti-CD16 antibody at a concentration of about 1.0 mcg/ml.
[0044] Most typically, the mononuclear cells have a density of 1-2 x 106
cells/ml in a total
volume of about 200 ml, and the cells and medium are in a single container.
After about 3-4
days, the cells are fed with fresh medium containing N-803, and further feed
cycles are
performed about every three days through recovery, rapid expansion, and
culture
culmination. Notably, successful NK cell expansion in such scheme was
significantly
dependent on the proper choice of stimulatory factors as is exemplarily shown
in Fig.2. Here,
a dramatic expansion of over 20,000-fold from day 0 was observed when using
anti-CD3 and
anti-CD16 monoclonal antibodies whereas anti-CD16 antibodies alone failed to
produce the
same dramatic effect. Notably, the presence of an anti-4-1BB antibody seemed
to
prematurely exhaust the proliferation of NK cells.
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[0045] Cells culture is then terminated upon reaching a desired quantity,
typically about 0.5-
5.0 x 109 total cells and/or upon reaching a desired expansion (e.g. at least
100-fold
expansion). Notably, despite the apparent simplicity, the so obtained cell
culture contains
after about three weeks more than about 85% NK cells, with less than about 8%
NKT cells,
and with less than about 2.5% T cells, and less than about 1.2% double
negative (DN) T cells.
Moreover, it should be recognized that the entire culture process may be
performed in a
single container within a self-contained bioreactor, which substantially
reduces risk of
contamination and eliminates reagent and cell handling during the cultivation
step.
[0046] Upon reaching a desired cell quantity, the cells can be transferred
into a fresh medium
for subsequent cytokine stimulation. Alternatively, the cytokine stimulation
to generate the
memory like phonotype can be performed in the same medium, typically by adding
further
medium with a stimulatory cytokine composition that includes an IL-18/IL-12-
TxM fusion
protein complex (or a mixture of IL-12, N-803, and IL-18, or a mixture of IL-
12, IL-15, and
IL-18). In most cases, the cytokine stimulation will be performed for a time
of between about
4-24 hours, and more typically between 12-16 hours. As will be readily
appreciated, the cells
can then be transferred into a transfusion medium prior to transfusion. In
addition, the
phenotype and/or cytotoxicity of the CIML NK cells may be determined and
exemplary
results are shown in more detail below.
[0047] With respect to suitable biological fluids, it is generally
contemplated that the fluids
can be autologous relative to the individual that will receive the NK cells
isolated in the
methods presented herein. Therefore, especially preferred biological fluids
include fresh
whole blood, cord blood (frozen or fresh), and cells separated in a
leukapheresis procedure.
However, it should be appreciated that the biological fluid may also be any
fluid that contains
NK cells (typically among other cell types). For example, suitable alternative
biological
fluids include whole blood from allogenic donors, which may or may not be
matched for a
compatible MHC type. Therefore, samples in a blood bank that approach
expiration date are
deemed suitable for use, as well as freshly donated whole or stored cord blood
by an
individual other than the NK cell recipient. Moreover, it should be noted that
where the
biological fluid is the cord blood, the cord blood may be matched and donated
after a
sufficient MHC match with the NK cell recipient. Likewise, it should be noted
that the
manner of isolating or enriching mononuclear cells may vary considerably, and
the person of
ordinary skill in the art will be readily apprised of the most suitable
methods of isolation and
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enrichment. For example, where the biological fluid is whole blood or cord
blood, it is
preferred that the fluid is processed via gradient density centrifugation
using any suitable
medium (e.g., Ficoll-Hypaque). Alternatively, mononuclear cells may be
obtained directly
from the patient by leukapheresis, or the biological fluid may be subjected to
removal of red
blood cells using antibodies. In still further methods, mononuclear cells may
be isolated using
magnetic bead separation where the beads are coated or otherwise coupled to
antibodies
binding the mononuclear cells.
[0048] Likewise, it should be recognized that the particular nature of the
medium for
activation and feeding need not be limited to NK MACS medium, but that all
media known
to support growth of NK cells are deemed suitable for use herein. Most
preferably, however,
defined media are used and may be supplemented with human AB serum.
[0049] Proliferation of the NK cells in the mixture of mononuclear cells is
preferably
stimulated and supported with a combination of an anti-CD16 antibody and N-
803, and
optionally an anti-CD3 antibody. There are various sources for anti-CD16
antibodies known
in the art/commercially available, and particularly preferred anti-CD16
antibodies have
agonist (activating) activity and are specific to human CD16. However,
activators other than
anti-CD16 antibodies are also deemed suitable for use herein include anti-CD16
antibody
fragments and fusion proteins with anti-CD16 antibody fragments. Additionally,
or
alternatively, contemplated activators also include CD314 or NKG2D, the
natural
cytotoxicity receptors CD335 (NKp46), CD336 (NKp44) and CD337 (NKp30), CD226
(DNAM-1), CD244 (2B4), members of the CD158 or killer immunoglobulin-like
receptor
(KIR) family that carry a short cytoplasmic tail (KIR2DS and KIR3DS) and
CD94/NKG2C,
among others.
[0050] Concentrations of the anti-CD16 antibody will typically follow those
already known
in the art for activation of NK cells. Therefore, suitable concentrations for
anti-CD16
antibodies will be between about 0.01-5.0 mcg/ml, and more typically between
about 0.01-
0.3 mcg/ml, or between about 0.05-0.5 mcg/ml, or between about 0.1-1.0 mcg/ml,
or
between about 1.0-5.0 mcg/ml. With respect to the duration of exposure to the
anti-CD16
antibody it is generally contemplated that the mixture of mononuclear cells is
exposed to only
a single, two, or there doses of the anti-CD16 antibody, most typically when
the mononuclear
cells are isolated and contacted with the activation medium for the first
(and/second, and/or
third) time. The person of ordinary skill in the art will be readily able to
recognize proper
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schedule and dosage to achieve NK cell activation. Most typically, exposure of
the
mononuclear cells to the anti-CD16 antibody is contemporaneous with exposure
of the
mononuclear cells with the N-803. However, in less preferred embodiments,
exposure of the
mononuclear cells to the anti-CD16 antibody is sequentially to exposure of the
mononuclear
cells with the N-803 (with exposure of the mononuclear cells to the anti-CD16
antibody first
being the preferred sequence).
[0051] Where desired, proliferation stimulation/support may also include
contacting the cells
with anti-CD3 antibody, typically at the same time of contacting the cells
with anti-CD16
antibody. As noted above, concentrations of the anti-CD3 antibody will
typically follow those
already known in the art for activation of NK cells. Therefore, suitable
concentrations for
anti-CD3 antibodies will be between about 0.01-10.0 ng/ml, and more typically
between
about 0.01-0.1 ng/ml, or between about 0.1-0.5 ng/ml, or between about 0.3-1.0
ng/ml, or
between about 1.0-5.0 ng/ml. Likewise, with respect to the duration of
exposure to the anti-
CD3 antibody it is generally contemplated that the mixture of mononuclear
cells is exposed
to only a single, two, or there doses of the anti-CD3 antibody, most typically
when the
mononuclear cells are isolated and contacted with the activation medium for
the first
(and/second, and/or third) time. The person of ordinary skill in the art will
be readily able to
recognize proper schedule and dosage to achieve NK cell activation.
[0052] With respect to N-803 it is contemplated that N-803 (an IL-15N72D:IL-
15RaSu/IgG1
Fc complex with human sequences; see US 2019/0023766, commercially available
from
ImmunityBio) is preferred as an agent in the activation and feed medium.
However, various
alternative agents with IL-15 activity are also deemed suitable for use
herein. In this context,
and without wishing to be bound by any theory or hypothesis, the inventors
contemplate that
N-803 enables growth and expansion of the NK cells by virtue of continuous
signaling. In
contrast, IL-15 as isolated cytokine has a very short lifespan and signaling
activity is typically
very short. This, where IL-15 as isolated cytokine is added to a growth
medium, the
signaling will be pulsed or intermittently. In contrast, where N-803 is
provided, stability of
IL-15 is dramatically extended and signaling is deemed continuous. Moreover,
it should be
recognized that N-803 also provides a physiological context (i.e., IL-15 R-
alpha chain) and a
N72D form that acts as a super agonist. Therefore, any stabilized IL-15
compound is also
expressly deemed suitable for use herein.
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[0053] For example, all compounds and complexes that effect IL-15 signaling
are deemed
suitable for use herein so long as such compounds and complexes have a serum
half-life that
is longer than isolated/recombinant and purified IL-15 alone. Moreover, it is
generally
preferred that the stabilized IL-15 compounds will include at least portions
of human
sequences for IL-15 and/or IL-15 Ra. For example, suitable compounds include
P22339 (a
complex of IL-15 and the Sushi domain of IL-15Ra chain with a disulfide bond
linking the
IL-15/Sushi domain complex with an IgG1 Fc to augment its half-life; see
Nature, Scientific
Reports (2018) 8:7675), and XmAb24306, which is a IL-15/IL-15Ra-Fc heterodimer
(see
e.g., WO 2018/071919).
[0054] In further especially contemplated embodiments, the mixture of
mononuclear cells is,
after isolation from the biological fluid, placed into a cell culture
container together with the
medium containing the anti-CD16 (and optionally anti-CD3) antibody and N-803
to activate
the NK cells. Most preferably, the container is a cell culture flask with at
least one wall (or
portion thereof) that is transparent to light such that cell shape, staining,
and/or growth can be
observed with a microscope or other optical instrument. Thus, it should be
noted that the cells
can be continuously or periodically monitored in a bioreactor, and so obtained
measurements
(e.g., cell size, cell number, cell distribution, etc.) can be used to trigger
or modify an
automated feeding schedule in a control unit that is logically coupled to the
bioreactor. Most
typically, and as shown in Fig. 2, feeding fresh medium with N-803 can be
performed using a
predefined schedule, typically every three days, where preferably each feeding
will include
N-803 to maintain continuous signaling. While the specific volumes in the
examples below
are suitable for expanding the NK cells to cell densities consistent with cell
growth, it should
be appreciated that the volumes may be adjusted to accommodate particular
growth patterns.
To that end, it should also be appreciated that the feeding may be
continuously or that
predetermined volumes may be changed in response to the growth kinetic
observed in the
container.
[0055] In most cases, the yield of the NK cells at the end of the cultivation
will be typically
at least 80%, or at least 82%, or at least 85%, or at least 88%, or at least
90%, or at least 92%,
or at least 94% of all live cells with the remainder being NKT cells, DN T
cells, and T cells.
For example, remaining NKT cells will typically be equal or less than 10%, or
equal or less
than 8%, or equal or less than 7%, or equal or less than 6% of all live cells,
while remaining
T cells will typically be equal or less than 5%, or equal or less than 4%, or
equal or less than
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3%, or equal or less than 2% of all live cells, and remaining DN T cells will
typically be
equal or less than 3%, or equal or less than 2%, or equal or less than 1.5 %,
or equal or less
than 1% of all live cells.
[0056] Therefore, and viewed from a different perspective, it should be
appreciated that the
systems and methods contemplated herein are capable of remarkably high
expansion of NK
cells, and typical expansions are at least 80-fold, or at least 100-fold, or
at least 120-fold, or
at least 130-fold, or at least 140-fold with respect to the number of NK cells
originally
present in the mixture of mononuclear cells. Such expansion is particularly
notable in view of
the very simple manner of activation and cultivating (one-pot process).
Indeed, once the
mixture of mononuclear cells is placed into the cell culture container, the
entire process can
continue within the same container and can be sustained by addition of media
only. Thus,
complex handling and expensive reagents are entirely avoided, and the risk for
contamination
is significantly reduced.
[0057] As already noted above, the NK cells can be expanded to a total cell
number of about
0.1- 1.0 x 109 cells, or about 0.3- 3.0 x 109 cells, or about 0.5- 5.0 x 109
cells, or about 0.7-
7.0 x 109 cells, or about 1- 10 x 109 cells, or even higher. The exact number
of expanded NK
cells will typically depend, among other things, on the particular purpose for
the NK cells,
culture conditions, and the starting number of cells. Upon reaching the
desired quantity of
cells, cytokine stimulation may then be performed in the expansion medium,
typically by
adding fresh medium that contains a stimulatory cytokine composition.
[0058] In most cases, the stimulatory cytokine composition will comprise one
or more
activating cytokines such as IL-2, IL-12, IL-15, IL-21, and to a lesser degree
also IL-4 and
IL-7. Of course, and as discussed in more detail below, suitable cytokines may
also be
derivatives of the above cytokines, and especially preferred derivatives
include fusion
complexes. Still further, it should be recognized that one or more of the
cytokines may also
be expressed in the expanded NK cells following transfection with an
appropriate
recombinant nucleic acid (e.g., transient expression from a plasmid or viral
expression
vector).
[0059] For example in some embodiments, the stimulatory cytokine composition
will
comprise an IL-18/IL-12-TxM fusion protein complex, and especially preferred
fusion
protein complexes are described in WO 2018/165208, which is incorporated by
reference
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herein. In such case, it should be appreciated that the fusion protein complex
provides three
cytokine functions (IL-12, IL-15, and IL-18) in a stabilized form via their
coupling to an Fc
portion of a human IgG. Moreover, while not wishing to be bound by any theory
or
hypothesis, the Fc portion of the fusion protein complex may provide a further
stimulatory
signal, possibly through interaction with CD16 on the expanded NK cells.
However, other
fusion protein complexes based on N-808 are also expressly contemplated
herein. For
example, suitable fusion protein complexes may include targeting scFv
portions, or cytokine
portions other than (or in addition to) IL-12 and IL-18. Of course, it should
be noted that
while an IL-18/IL-12-TxM fusion protein complex is in many cases preferred,
alternative
TxM fusion protein complexes are also deemed suitable and especially
contemplated fusion
complexes will include a IL15/IL-15Ralpha portion as described in WO
2018/165208, and at
least one additional cytokine selected from the group consisting of IL-7, IL-
18, and IL-21.
Therefore, and among other suitable choices, contemplated TxM fusion complexes
include an
IL-18/IL-7 TxM and/or IL-18/IL-21 TxM.
[0060] Therefore, in other examples, the stimulatory cytokine composition may
also
comprise a derivative of IL-15, and especially preferred derivatives are those
based on N-
803. Such derivatives will advantageously have increased signaling effect as
compared to IL-
15 per se due to the presence of the IL-15Ra chain, and exemplary suitable
derivatives are
described in WO 2016/004060 and WO 2018/075989. Most typically, where N-803 or
similar
fusion proteins are used, additional cytokine functions will be supplied by
individual
cytokines, and especially IL-7, IL-12, IL-21, and IL-18. Therefore, in yet
another aspect of
the inventive subject matter, the stimulatory cytokine composition may also
comprise IL-7,
IL12, IL-15, IL-21, and IL-18 as individual cytokines. Therefore, and among
other choices,
such individual cytokines may be added alone or in combination with other
individual
cytokines or TxM constructs, each or which may be recombinant (or even
recombinantly
expressed in the cell).
[0061] Thus, it should be appreciated that one or more of the stimulatory
cytokines can also
be (temporarily) expressed from a recombinant nucleic acid that is transfected
into the
expanded NK cells. For example, suitable transfection methods include viral
transfection
where the recombinant nucleic acid is a viral expression vector. On the other
hand, the
recombinant nucleic acid may also be transfected into the cell using
electroporation or
lipofection using methods well known in the art. Furthermore, where
electroporation or
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lipofection is employed, it is typically preferred that the nucleic acid is an
RNA (however,
DNA is also deemed suitable for use herein).
[0062] Regardless of the particular type of stimulatory cytokine composition,
it is generally
contemplated that the cytokine or cytokines are present in the medium at a
concentration
effective to generate a memory like phenotype of the NK cell. Therefore,
suitable total
cytokine concentrations will be between 0.1 nM and 1.0 nM, or between 0.5 nM
and 5.0 nM,
or between 1.0 nM and 10 nM, or between 10 nM and 50 nM, and in some cases
even higher.
Where multiple cytokines are used, it is generally preferred that the
cytokines are present in
substantially equimolar concentrations (+/- 50% deviation). On the other hand,
where the
stimulatory cytokine composition comprises an IL-18/IL-12-TxM fusion protein
complex, the
complex may be present between 0.5 nM and 5.0 nM, or between 1.0 nM and 10 nM,
or
between 10 nM and 50 nM, or even higher.
[0063] With respect to the timing of the stimulatory cytokine composition it
is generally
preferred that the NK cells are first expanded to a desired (typically final)
quantity prior to
exposure to the stimulatory cytokine composition. However, in alternative
aspects, the
stimulatory cytokine composition can be added to the expanding NK cell
population starting
at about 70% of the final desired cell quantity, or starting at about 80% of
the final desired
cell quantity, or starting at about 90% of the final desired cell quantity. In
most aspects of the
inventive subject matter, the exposure to the stimulatory composition will
last be between
about 2 hours and 48 hours, or between 4 hours and 8 hours, or between 8 hours
and 12
hours, or between 12 hours and 24 hours, and in some cases even longer.
[0064] Exposure to the stimulatory cytokine composition can be terminated by
replacement
of the medium, typically with fresh medium or a medium suitable for
transfusion. On the
other hand, it is also contemplated that the so generated CIML NK cells can be
subjected to a
resting period prior to subsequent use that can last that between 0-4 hours,
between 4-12
hours, between 12 and 24 hours, or between 1-4 days, and even longer. As will
also be
readily appreciated, the CIML NK cells may also be subjected to re-stimulation
to further
increase cytotoxicity, and re-stimulation will typically be performed using at
least one
stimulatory cytokine such as IL2 or IL-15. Most preferably, and as is shown in
more detail
below, re-stimulation provided unexpectedly high cytotoxicity where N-803 was
used (as
compared to IL-15 per se). Moreover, it should be noted that re-stimulation
will typically
follow standard protocols well known in the art.
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[0065] Regardless of the final treatment of the CIML NK cells, it is
contemplated that the
CIML NK cells will be used for transfusion to an individual in need thereof,
and most
typically, the individual will be diagnosed with a cancer. As will also be
readily appreciated,
the CIML NK cells may form of a treatment regimen in which the individual
receives a
cancer vaccine (e.g., recombinant (adeno)viral vaccine, recombinant yeast
vaccine,
recombinant bacterial vaccine), a chemotherapeutic agent, a checkpoint
inhibitor, N-803 or a
TxM-based therapeutic, and/or a targeted interleukin (e.g., NHS-IL12).
[0066] While not limiting to the inventive subject matter, it is further
contemplated that the
CIML NK cells are expanded and/or activated in a culture environment that
allows for
continuous monitoring, continuous management of CO2 and 02 levels, and
continuous
monitoring to detect cell density (e.g., confluence). Among other options for
such
environments, especially preferred environments are automated cell culturing
and harvesting
devices as are described, for example, in WO 2015/165700. Such `GMB-in-a-box'
systems
beneficially allow control over feeding schedules, gas control, allow for real-
time detection of
cell density, growth (kinetics) and cell health, as well as dramatically
reduce the possibility
of contamination due to significantly reduced handling requirements.
[0067] In still further contemplated aspects, it should be noted that the
systems and methods
presented herein advantageously also allow generation of CD56d1m and CD 56bri
ght NK cells,
particularly where the NK cells are generated from peripheral blood. Depending
on further
culture conditions, CD56bright NK cells may then differentiate to CD56dim
cells. Such distinct
NK cell populations can then be employed as for distinct therapeutic options
due to their
distinct maturation and cytotoxicity profile. Additionally, it should be
appreciated that the
compositions, systems and methods will also be suitable to generate NKT cells
upon proper
stimulation and culture.
Examples
[0068] In view of the above, and as provided in more detail below, one
exemplary method
entailed isolating CBMCs or PBMCs by a single Ficoll centrifugation step,
which was
followed by incubation of the cells with about 0.4 nM N-803 and about 0.1
mcg/ml of an
anti-CD16 antibody (e.g., clone B73.1, commercially available from BD
Biosciences), and
optionally about 0.5 ng/ml of an anti-CD3 antibody in NK MACS media with 10%
human
AB serum. Typically 100-150 mL (typically 135 mL) of CBMCs at a million
cells/ml were
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used as the starting material with above reagents. Media was used for dilution
with N-803
twice a week (3-5 day intervals) with a regimen of a 1:2 and 1:10 compared to
existing
volume with corresponding concentration of N-803 for a final concentration of
0.4 nM. The
expansion culture is typically terminated when the expanded NK cells make up
about
between 90% and 99% (e.g., 98%) of all cells. Upon termination, cytokine
induction can be
performed as described in more detail below.
[0069] MNCs were freshly isolated from cord blood or peripheral blood. It was
washed twice
with complete NKMACS medium (NKMACS+ Supplements+ 10% hu-AB-serum). MNCs
were suspended in 150mL of medium with density of 1x10^6 cell/mL in a GMP box
(500mL
volume). 150mL cell suspension was supplemented with anti-CD16 antibody
(1mcg/mL) and
N-803 (0.4nM). GMP Box started imaging and cells were propagated according to
pre-
programmed steps. Cells in the GMP box were supplemented with 10X cytokine
medium or
with 2X cytokine medium in alternate fashion. NK enrichment (phenotype for
CD3, CD56,
and CD16 expression) and cell health (cell number, viability, and cell
density) were
monitored regularly and plotted.
[0070] Cytokine induction to generate CIML NK cells from expanded NK cells was
started
upon reaching a point at which 98% of all cells were NK cells. To that end, a
box with
500mL and 2.3x10^6 cells/mL density was equally split into two separate boxes.
Thus,
500mL cell suspension became 250mL in two the respective boxes and the cells
were diluted
1:1 with fresh medium. Subsequently, IL18/12 TxM was added to a final
concentration of 10
nM (for control and comparison, N-803 was used at a final concentration of
0.07 nM) and the
cells were incubated with the IL-18/IL-12-TxM fusion protein complex for 16
hours to so
obtain the CIML NK cells. For further testing, the cells were washed and then
subjected to
expression analyses and cytotoxicity assays.
[0071] Materials: MNCs from Cord and Peripheral Blood, anti-CD16 antibody, BD
bioscience San Diego CA; NK MACS medium with NK supplement, staining
antibodies for
phenotyping (aCD3, aCD16, aCD56, aNKp30, aNKp44, aNKp46, aNKG2A, aNKG2D,
aTIGIT, aCD34, aTRAIL, aCD57, aCXCR3, and aCCR5), Miltenyi Biotec San Diego,
CA;
Human AB serum, Access Biologicals, San Diego CA; N-803, GMP in a Box kit,
Nantbio
Inc. Culver City CA. IL-18/IL-12-TxM fusion protein complex was obtained from
ImmunityBio.
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[0072] So generated CIML NK cells were tested for cytotoxicity and selected
surface marker
expression. More particularly, in one set of experiments cord blood derived
CIML NK cells
were tested against Merkel cell carcinoma cells (here MS-1 cells) that are
typically resistant
to NK cytotoxicity. Notably, and as can be seen in Fig.3, the CIML NK cells
had significant
cytotoxicity after expansion and control exposure to the IL-18/IL-12-TxM
fusion protein
complex, whereas some cytotoxicity was even observed when the cord blood cells
were
exposed to N-803 only. Fig.4 depicts exemplary results for surface marker
expression in cord
blood derived cells exposed to the IL-18/IL-12-TxM fusion protein complex and
N-803. As
can be seen, the CIML NK cells had reduced expression of CD16, but
substantially increased
expression of CD25, DNAM1, and strong secretion of IFN-y.
[0073] Similar results were obtained when the CIML NK cells were derived from
peripheral
blood as can be seen in Fig.5. Here, CIML NK cells had substantial
cytotoxicity against the
MS-1 cell line, and the N-803 control cells from peripheral blood also showed
some
cytotoxicity. Likewise, the surface markers for the peripheral blood derived
CIML NK cells
showed decreased expression of CD16 and TIGIT, while having significant
increases in
CD25, DNAM1, and IFN-y secretion as can be taken from Fig.6. Notably, when NK
cells
were cultivated using standard cultivation protocols or where fresh NK cells
were used, no
significant cytotoxicity against MS-1 cells were observed, even where the
cells were induced
with IL-12, IL-15, and IL-18 to trigger a memory like phenotype.
[0074] Cord blood derived CIML NK cells were also tested for the activation
cluster
phenotype and Fig.7 depicts exemplary results comparing control exposure with
N-803 with
exposure to the IL-18/IL-12-TxM fusion protein complex. As can be seen from
the images,
there is a striking difference in the culture morphology after overnight
exposure to the IL-
18/IL-12-TxM fusion protein complex versus exposure to N-803. When looking at
selected
surface markers of these CIML NK cells, it was yet again apparent that
exposure to the IL-
18/IL-12-TxM fusion protein complex resulted in a significant increase of CD25
(which is a
known activation associated receptor) as shown in Fig.8. Clearly, cytokine
stimulation with
IL-12, IL-15, IL-18 functions substantially increased the CD25 presentation,
which is
typically not observed (at least to that degree) with conventional fresh NK
cells.
[0075] Such increase in activating receptors and decrease in inhibitory
receptors was also
readily evident when observing culture morphology in a kill assay on K562
cells as is shown
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in Fig.9. Here, the cord blood derived CIML cells upon re-stimulation
exhibited substantially
increased activation clustering as compared to incubation with N-803.
[0076] In further experiments, the inventors also investigated the time course
of cytotoxicity
on K562 cells as is exemplarily depicted in Fig.10A, Fig.10B, and Fig.10C
showing results
after 24 hours, 48 hours, and 72 hours, respectively. After the first 24 hour
time-point
(Fig.10A) one can see the start of increased killing capacity on K562 cells as
both TxM
concentrations tested have lower EC50s than the N-803 control. At 48 hours
(Fig. 10B)
increased killing by the TxM treated samples is seen, but less so for the N-
803 control. At
this time-point the increase in killing of K562 is about 3-fold. At 72 hours
(Fig.10C) all
conditions have begun to lose activity on K562 killing, but the TxM treated
cells retain their
enhanced killing by about 3-fold compared to the control.
[0077] Fig.11 provides a direct comparison for expanded cord blood derived NK
cells,
expanded cord blood derived NK cells with N-803 stimulation, and expanded cord
blood
derived NK cells stimulated with the IL-18/IL-12-TxM fusion protein complex
for 24 hours.
As can be seen, the cytotoxicity for all cells is readily evident, with
expanded NK cells
having a slight advantage over %max kill, but requiring a substantially higher
E;T ratio as
compared to the CIML cells. Fig.12 depicts expression of selected markers of
expanded
peripheral blood derived NK cells with N-803 stimulation versus expanded
peripheral blood
derived NK cells stimulated with the IL-18/IL-12-TxM fusion protein complex.
As can be
taken from Fig.12, there is a significant downregulation of TIGIT (and CD16)
and a
significant upregulation of CD25, which is indicative of activation. It should
be noted that
the downregulation of CD16 may be accompanied by a reduction in ADCC. However,
the
potential reduction in ADCC is outbalanced by the higher activation and
cytotoxicity against
cell lines that would otherwise be resistant to NK cell cytotoxicity. Similar
cytotoxicity
results are found with peripheral blood derived CIML NK cells after 24 hours
stimulation
with an IL-18/IL-12-TxM fusion protein complex as is shown in Fig.13. Clearly,
exposure to
nM of the IL-18/IL-12-TxM fusion protein complex generated better cell killing
in the
1(562 assay.
[0078] Secretion of IFN-y was tested for peripheral blood derived CIML NK
cells and Fig.14
shows exemplary results using different conditions. The same cells were also
used in a
cytotoxicity assay and Fig.15 shows exemplary results. Similar results are
provided for cord
blood derived CIML NK cells as can be taken from Fig.16. Notably, cytokine
induction with
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N-803 outperformed induction with IL-15 per se. Thus, it should be noted that
while multi-
cytokine induction is preferred as shown above, induction with N-803 is also
expressly
contemplated.
[0079] As used herein, the term "administering" a pharmaceutical composition
or drug refers
to both direct and indirect administration of the pharmaceutical composition
or drug, wherein
direct administration of the pharmaceutical composition or drug is typically
performed by a
health care professional (e.g., physician, nurse, etc.), and wherein indirect
administration
includes a step of providing or making available the pharmaceutical
composition or drug to
the health care professional for direct administration (e.g., via injection,
infusion, oral
delivery, topical delivery, etc.). Most preferably, the cells or exosomes are
administered via
subcutaneous or subdermal injection. However, in other contemplated aspects,
administration
may also be intravenous injection. Alternatively, or additionally, antigen
presenting cells may
be isolated or grown from cells of the patient, infected in vitro, and then
transfused to the
patient. Therefore, it should be appreciated that contemplated systems and
methods can be
considered a complete drug discovery system (e.g., drug discovery, treatment
protocol,
validation, etc.) for highly personalized cancer treatment.
[0080] The recitation of ranges of values herein is merely intended to serve
as a shorthand
method of referring individually to each separate value falling within the
range. Unless
otherwise indicated herein, each individual value is incorporated into the
specification as if it
were individually recited herein. All methods described herein can be
performed in any
suitable order unless otherwise indicated herein or otherwise clearly
contradicted by context.
The use of any and all examples, or exemplary language (e.g., "such as")
provided with
respect to certain embodiments herein is intended merely to better illuminate
the the full
scope of the present disclosure, and does not pose a limitation on the scope
of the invention
otherwise claimed. No language in the specification should be construed as
indicating any
non-claimed element essential to the practice of the claimed invention.
[0081] It should be apparent to those skilled in the art that many more
modifications besides
those already described are possible without departing from the full scope of
the concepts
disclosed herein. The disclosed subject matter, therefore, is not to be
restricted except in the
scope of the appended claims. Moreover, in interpreting both the specification
and the claims,
all terms should be interpreted in the broadest possible manner consistent
with the context. In
particular, the terms "comprises" and "comprising" should be interpreted as
referring to
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elements, components, or steps in a non-exclusive manner, indicating that the
referenced
elements, components, or steps may be present, or utilized, or combined with
other elements,
components, or steps that are not expressly referenced. Where the
specification claims refers
to at least one of something selected from the group consisting of A, B, C
.... and N, the text
should be interpreted as requiring only one element from the group, not A plus
N, or B plus
N, etc.
22
