Note: Descriptions are shown in the official language in which they were submitted.
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EX VIVO METHODS FOR MINIMIZING RISKS AND MAXIMIZING BENEFITS OF
ALLOGENEIC BLOOD AND MARROW TRANSPLANTATION
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
provisional patent
application serial number 62/101,181, filed January 8, 2015, entitiled "EX
VIVO METHODS
FOR MINIMIZING RISKS AND MAXIMIZING BENEFITS OF ALLOGENEIC BLOOD
AND MARROW TRANSPLANTATION", the content of which is incorporated by reference
herein in its entirety.
FIELD OF INVENTION
[0002] The described invention relates to allogeneic blood and bone
marrow
transplantation (BMT), ex vivo approaches to minimizing gastrointestinal graft
versus host
disease prior to BMT, and ex vivo approaches to maximizing graft versus tumor
effects.
BACKGROUND OF THE INVENTION
Graft-versus-Host Disease (GVHD) Screening and Prevention
[0003] Allogeneic blood and marrow transplantation (allo-BMT) is an
effective
immunotherapeutic treatment that can provide partial or complete remission for
patients with
drug-resistant hematological malignancies, including acute myeloid leukemia,
lymphoma,
and multiple myeloma.
[0004] According to the Center for International Blood & Marrow
Transplant
Research, 7,000 allo-BMT procedures were performed in the U.S. in 2011
(Pasquini MC,
Current use and outcome of hematopoietic stem cell transplantation: CIBMTR
Summary
Slides, <http://www .cibmtr.org> (2013)). In this procedure, mature donor T
cells in the
donor inoculum play a central role in mediating graft-versus-tumor (GVT)
responses by
destroying residual tumor cells that persist after conditioning regimens.
However, the full
exploitation of this clinical intervention has been greatly limited by the
development of graft-
versus-host disease (GVHD), a complication that can occur after a stem cell or
bone marrow
transplant that is caused by donor T cell recognition of alloantigens
expressed on the patient's
tissue cells, particularly in the lymphoid compartment, intestine, skin, and
liver (Ferrara, et
al., Lancet (2009) 373, 1550-1561; Sung, et al., Clinical Haematology (2013)
26, 285-292;
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Sung, et al., Stem Cells Translational Medicine (2013) 2, 25-32). Even in
fully human
leukocyte antigen (HLA)-matched transplant settings, alloreactivity towards
minor
histocompatibility antigens (miHA), which are self peptides presented by HLA
molecules,
drives both the development of GVT and GVHD effects. However, clinical
dissociation and
regulation of these two responses to improve transplant outcomes have not yet
been achieved.
As a result, the global incidence of acute GVHD ranges from 26-34% in fully
HLA-matched
sibling recipients and 42-52% in HLA-matched, unrelated recipients (Sung, et
al., Clinical
haematology (2013) 26, 285-292; Jacobs, et al., Bone marrow transplantation
(2012) 47,
1470-1473; Jagasia, et al., Blood (2012) 119, 296-307). GVHD of the
gastrointestinal (GI)
tract is closely associated with non-relapse mortality following allo-BMT
(Harris, et al.,
Clinical Haematology (2012) 25, 473-478).
[0005] Consistent with a 2005 National Institutes of Health (NIH)
Consensus
Conference, classification of GVHD is based on clinical presentation rather
than time of
onset. (Pidala, J. et al., "Overlap subtype of chronic graft-versus-host
disease is associated
with an adverse prognosis, functional impairment, and inferior patient-
reported outcomes: a
Chronic Graft-versus-host Disease Consortium Study, Haematologica 97(3): 451-
458
(2012)).
[0006] Acute GVHD manifestations include erythematosus or
maculopapular
rash, nausea and vomiting or diarrhea and cholestatic hepatitis, and
historically was limited to
within 100 days following HCT. Grading for acute GVHD divides acute GVHD into
four
stages based on the extent of involvement of the skin, liver and
gastrointestinal tract. In stage
I, there is a skin rash over <25% of the body, bilirubin is measured at 26-60
mon, with a
gut fluid loss 500-1000mL/day. In stage II, a skin rash covers 25-50% of the
body, the
bilirubin is measured at 61-137 mon, and the gut loses from 1000-1500 mL/day.
Stage III
is characterized by involving >50% of the skin, the bilirubin is measured at
138-257mol/L,
and the gut has lost more than 1500 mL/day. Stage IV is characterized by
bullae
desquamation (blisters with shedding of epidermal cells) of skin, the
bilirubin exceeds
>257mol/L, and the gut fluid loss is >2500 mL/day or ileus (disruption of the
normal
propulsive ability of the gastrointestinal tract; bowel obstruction).
[0007] In acute GVHD, histological tissue damage can be observed as
early as 8
days in HLA-matched recipients and sometimes as late as one to two months
after BMT.
(MacMillan, M.L. et al., "What Predicts high risk acute graft-versus host
disease (GVHD) at
onset?: identification of those at highest risk by a novel acute GVHD risk
score. Br. J.
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Haematol. 157: 732-41, doi: 10.111/j.1365-2141.2012.09114.x (2012); Kambham,
N. et al.,
"Hematopoietic stem cell tyransplantation: graft versus host disease and
pathology of
gastrointestinal tract, liver, and lung," Adv. Anat. Pathol. 21: 301-320, doi:
10.1097/PAP.0000000000000032 (2014)). The relatively slow progression of GVHD
is due
to the presence of low frequency responses against minor histocompatibility
antigen (miHA)
differences that are present in HLA-matched settings and drive the
alloreactive response.
Zilberberg, J. et al, "Inter-strain tissue-infiltrating T cell responses to
minor
histocompatibility antigens involved in graft-versus-host disease as
determined by Vbeta
spectratype analysis," J. Immunol. 180: 5352-59 (2008); Berger, M. et al, T
cell subsets
involved in lethal graft-versus host disease directed to immunodominant minor
histocompatibility antigens," Transplantation 557: 1095-1102 (1994); Korngold,
R. &
Sprent, J., "Lethal graft-versus host disease across minor histocompatibility
barriers in mice,"
Clin. Haematol. 12: 681-693 (1983)). In vivo, T-cell frequencies to miHA have
been
observed on the order of 1 in 300,000 cells per target, but it can be
increased to 1 in 5,000-
8,000 upon activation and expansion of the specific T cell clones. The, H.S.
et al., Selection
of the T cell repertoire during ontogeny: limiting dilution aanalysis," Eur.
J. Immunol. 12:
887-892, doi: 10.1002/eji.1830121016 (1982); Simon M. M. & Eichmann, K.,
"Limiting
dilution analysis of alloreactive T helper cells: precursor frequencies
similar to that of
alloreactive cytotoxic T cells," Immunobiol. 164: 78-89, doi: 10.1016/S0171-
2985(83)80020-5 (1983)). In a graft-versus leukemia murine model, exposure to
allogeneic
tumor miHA significantly increased tumor-specific T-cell frequency and
significantly
decreased the number of T cells required to abrogate tumor burden. (Fanning,
S.L., et al.,
"Unraveling graft-versus-host disease and graft-versus-leukemia responses
using TCR Vbeta
spectratype analysis in a murine bone marrow transplantation model." J.
Immunol. 190:
447-57, doi: 10.4049/jimmuno1.1201641 (2013)). However, conventional in vitro
static
activation of primary human T cells lacks the capability of providing these
persistent
stimulatory events found in vivo, because APCs are often killed by donor T
cells or are
otherwise exhausted. In contrast, lymph nodes act in vivo as a site for
continued activation,
proliferation, and differentiation for effector T cells that can then migrate
to target tissues.
[0008] Acute GVHD manifestations occurring more than 100 days after
hematopoietic cell transplantation are classified as "persistent",
"recurrent", or "late onset"
acute GVHD, depending on the antecedent history of acute GVHD and absence of
other
chronic GVHD manifestations. Id.
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[0009] Classic chronic GVHD, which can result in multiple clinical
features
involving multiple sites (eyes, gastrointestinal tract, liver, lungs, heart,
bone marrow and
kidneys), is defined by diagnostic manifestations of chronic GVHD without
characteristic
features of acute GVHD, with extensive skin involvement, elevated bilirubin,
gastrointestinal
tract involvement and progressive onset from acute GVHD as poor prognostic
findings.
(Pidala, J., "Overlap subtype of chronic graft-versus-host disease is
associated with an
adverse prognosis, functional impairment, and inferior patient-reported
outcomes: a Chronic
Graft-versus-Host Disease Consortium study," Haematologica 97(3): 451-458
(2012)).
[0010] An overlap subtype of GVHD, which displays features of both
chronic and
acute GVHD, is a condition with an adverse prognosis, functional impairment,
and
significantly higher symptom burden. Patients with acute features have
significantly higher
non-relapse mortality and lower overall survival. These patients suffer
significant and
diverse functional impairments compared to those with classic chronic GVHD,
suggesting a
systemic functional impairment beyond the more direct ramifications of
concurrent acute
GVHD manifestations. Id.
[0011] There is a very strong correlation between GI GVHD severity
with
patients as well as with experimental outcomes. (Harris, A.C., Levine, J.E., &
Ferrara, J.L.
"Have we made progress in the treatment of GVHD? Best Pract. Res. Clin.
Haematol. 25:
473-78, doi: 10.1016/j.beha.2012.10.010 (2012); Fanning, S. L. et al.,
"Unraveling graft-
versus-host disease and graft-versus-leukemia responses using TCR Vbeta
spectratype
analysis in a murine bone marrow transplantation model." J. Immunol. 190: 447-
57, doi:
10.4049/jimmuno1.1201641 (2013); Zilberberg, et al., Inter-strain tissue-
infiltrating T cell
responses to minor histocompatibility antigens involved in graft-versus host
disease as
determined by Vbeta spectratype analysis. J. Immunol. 180: 5352-59 (2008). Two
independent investigations on risk factors for developing acute GVHD by
sampling cohorts
of more than 5,000 (6) and 864 (19) patients, found that (1) only 1-3%
experienced liver
GVHD of any grade; (2) 17-21% had skin + gastrointestinal (GI) GVHD; (3) 56%
developed
skin GVHD grades II-1V (apparent without biopsy); and (4) 17% had GI GVHD
grades II-
1V.
[0012] One of the major determinants for development and severity of
acute
GVHD in human transplantation is disparity in major and minor
histocompatibility antigens,
with an increasing number of mismatched antigens predicting greater risk of
acute GVHD
and nonrelapse mortality. (Goulmy, E et al, "Mismatches of minor
histocompatibility
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antigens between HLA-identical donors and recipients and the development of
graft-versus-
host disease after bone marrow transplantation," N. Eng. J. Med. (1996) 334
(5): 281-285;
Lee, SJ et al., "High resolution donor recipient HLA matching contributes to
the success of
unrelated donor marrow transplantation. Blood (2007) 110 (13): 4576-83).
Polymorphism
in non-HLA genes, including cytokines such as tumor necrosis factor (TNF),
interleukin 10
(IL-10), interferon gamma, KIR polymorphism, and NOD2/CARD15 gene
polymorphism,
also may contribute to the development and severity of acute GVHD. (J. Pidala,
"Graft-vs-
Host Disease Following Allogeneic Hematopoietic Cell Transplantation" (2011)
Cancer
Control; 18(4): 268-276, at 269).
[0013] There are several hypotheses as to mechanisms of chronic GVHD
pathogenesis: (1) thymic damage, in part mediated by prior acute GVHD, may
impair the
process of negative selection by thymic medullary epithelial cells that
eliminate pathogenic T
cells responsible for immunity; (2) the potential role of transforming growth
factor-beta has
been supported by amelioration of chronic GVHD manifestations after
neutralization of this
cytokine in murine models, and the clinical observation of an inverse
relationship between
transforming growth factor-beta signaling in CD4 and CD8 cells and the risk of
chronic
GVHD; and (3) B cells may play a role in chronic GVHD pathogenesis. (Id. at
271).
[0014] A conceptual model for GVHD suggests that GVHD is composed of
phases that include tissue damage from conditioning therapy and activation of
antigen-
presenting cells, activation of donor T cells resulting in differentiation and
migration, and
finally an effector phase in which host tissue damage is mediated by
inflammatory cytokines,
such as TNF-a and IL-1, and effector cells, most notably cytotoxic T cells.
Pidala, J., "Graft-
vs-Host Disease Following Allogeneic Hematopoietic Cell Transplantation,"
Cancer Control
18(4): 268-276 (2011). It is additionally complicated by disturbances in
pathways of
immunological reconstitution and failure to acquire immunological tolerance,
thereby
resulting in both alloimmune and autoimmune attacks on multiple host tissues.
(S.Z. Pavletic
and D.H. Fowler, "Are we making progress in GVHD prophylaxis and treatment?"
Hematology: Am. Soc. Hematol. Educ. Program (2012); 2012: 251-264.)
[0015] The process of donor alloreactive T cell migration and
infiltration in the
body starts with activation of these cells in secondary lymphoid tissues,
where they encounter
antigen presenting cells (APCs). During the first 2-3 days post BMT,
alloreactive T cells
recognize their cognitive alloantigens on APCs and both cell types produce and
respond to
proinflammatory cytokines, such as IFN-y and TNF-a. A complex cascade of
events enables
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the activated/effector T cells, which have upregulated surface expression of
chemokine
receptors and integrins, to migrate to the inflamed tissue sites where they
continue to see
alloantigen and induce reactivity against targeted epithelial tissues. Clark,
R.A. "Skin-
resident T cells: the ups and downs of on site immunity. J. Invest. Dermatol.
130: 362-370,
doi: 10.1038/jid.2009.247 (2010); Farber, D.L., et al, "Human memory T Cells:
generation,
compartmentalization and homeostasis," Nature Rev. Immunol. 14: 24-35, doi:
10.1038/nri3567 (2014); Wysocki, C.A. et al, Leukocyte migration and graft-
versus-host
disease," Blood 105: 4191-99, doi: 10.1182/blood-2004-12-4726 (2005). The
activated/effector T cells then re-enter the circulatory system and via
upregulated adhesion
molecules migrate to target organs where they mediate the pathologic damage to
tissues
resulting in GVHD. Ferrara, J. L. et al, "Graft versus host disease," Lancet
373: 1550-61,
doi: 10.1016/S0140-6736 (009)60237-3 (2009); Wysocki, C.A. et al., "Leukocyte
migration
and graft versus host disease," Blood 105: 4191-99, doi: 10.1182/blood-2004-12-
4726
(2005); Korngold, R. and Antin, JH, "Biology and management of acute graft
versus host
disease," Cancer Treat. Res. 144: 257-75, doi: 1O.1007/978-O-387-78580-6_11
(2009).
[0016] Approaches to decrease GVHD incidence after allo-BMT have
focused
mainly on suppression or deletion of donor T cells by broadly acting agents or
via cell
separation techniques. These methods lack specificity and increase the risk of
complications,
including: (1) slow immune reconstitution and subsequent opportunistic
infections; and (2)
more critically, tumor relapse.
[0017] Although fatal GVHD, manifesting as acute or chronic
inflammatory
destruction of the gut, lungs, skin, and other organs, can be completely
abrogated in animals
and humans by careful depletion of mature lymphocytes from the donor bone
marrow graft,
when this approach has been taken in patients being treated for various
cancers, the incidence
of tumor relapse is greatly increased, due to the loss of a graft vs. tumor
(GVT) effect, which
is characterized by an immune response to a graft recipient's tumor cells by a
donor's
transplanted immune cells in the bone marrow or peripheral blood. Donor immune
cells that
have been implicated in the GVT effect include CD4+ T cells, CD8+ T cells and
natural
killer (NK) cells. These cells are believed to use Fas-dependent killing and
perforin
degranulation to eradicate malignant cells. In addition to immune cells,
cytokines such as
interleukin-2 (IL-2), interferon-y (IFN-y) and tumor necrosis factor-a (TNF-a)
have been
shown to potentiate the GVT effect. (Ringden, O. et al., "The allogenic graft-
versus-cancer
effect", Brit.Haematol. (2009); doi:10.1111/j.1365-2141.2009.07866.x).
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[0018] Currently, there is no clinically viable means of assessing
which HLA-
matched patients will develop GVHD, and particularly in the GI tract, prior to
BMT.
Significant technical challenges associated with expanding primary human IECs
ex vivo and
with finding an IEC-T cell co-culture system that enables alloreactive
responses has made
such assessment difficult, if not impossible.
[0019] Although microphysiologically relevant human 3D tissue and
tumor
models cannot replicate the biological and physiological complexity associated
with
homeostatic and disease progressions that occur over a long period of time,
such models may
provide "snapshot" ex vivo reproductions of authentic phenotypic cell
functions and
interactions relating to specific persons and disease states.
[0020] It is well recognized that serially cultured human diploid
cells have a finite
lifetime in vitro. Hayflick, L. Exptl Cell Res. 37: 614-636 (1965). After a
period of active
multiplication, generally less than one year, these cells demonstrate an
increased generation
time, gradual cessation of mitotic activity, accumulation of cellular debris,
and, ultimately,
total degeneration of the culture. Id. However, conventional practices of
immortalizing
human cells into cell lines by gene transfection perturbs the cells' gene
expression profiles,
cellular physiology and physical integrity of their genome. Even if primary
cells can be
grown and maintained, gene expression and cellular physiology of such cells
can be
fundamentally different in 2D versus 3D culture environments.
[0021] The described invention provides a multiwell plate-based
perfusion tissue
cell culture device that is engineered to mimic the persistent stimulatory
events encountered
by circulating T cells at the lymph node and tissue levels much like it occurs
in a patient's
body.
[0022] This biomimetic approach is a departure from current in vitro
cultures in
which donor T cells are activated solely with patient-derived peripheral blood
lymphocytes
(Friedman, et al. Blood (2008) 112, 3517- 3525; Jarvis, et al. J.Clin. Path.
(2002) 55, 127-
132).
[0023] The described invention provides an ex vivo model of persistent
T
lymphocyte stimulation events encountered by circulating T lymphocytes at
lymph node and
tissue levels in vivo; methods for optimizing donor selection for allo-BMT,
methods for
assessing a patient's risk of developing GVHD before allo-BMT, which can help
in
optimizing donor selection, methods for measuring the potential killing of
patient-derived
intestinal epithelial cells (IECs) by donor T cells, where IECs are the
primary population
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targeted in GI GVHD (Hanash, et al., Immunity (2012) 37, 339-350; El-Asady, et
al., J. Exp.
Med. (2005) 201, 1647-1657), and methods to minimize risk of GVHD before allo-
BMT and
to maximize GVT effects in allo-BMT.
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SUMMARY OF THE INVENTION
[0024] According to one aspect, the described invention provides an ex
vivo
model of persistent T lymphocyte stimulation events encountered by circulating
T
lymphocytes at lymph node and tissue levels in vivo comprising (a) a multiwell
plate-based
perfusion culture device, comprising, from top to bottom: a bottomless multi-
well plate
comprising a plurality of bottomless wells; a first micropatterned polymer
layer attached to a
bottom surface of the bottomless multi-well plate to form a plurality of
adjacent wells, one or
more of each pair of adjacent wells comprising a transparent polymer membrane
placed
within the one ore mover of each pair of adjacent wells; a second
micropatterned polymer
layer comprising two or more holes that correspond to two or more adjacent
wells, the second
micropatterned polymer layer being attached to a bottom surface of the first
micropatterned
polymer layer, such that each hole of the second micropatterned polymer layer
is aligned with
the two or more adjacent wells in the first micropatterned polymer layer, one
or more of each
pair of adjacent wells comprising the transparent polymer membrane; a
microfluidic channel
formed between the two adjacent wells that allows internal fluidic
communication between
the two adjacent wells; one or more removable polymer plugs, each located at a
top surface
of each of the plurality of wells, and one or more tubes, each connected to
the one or more
polymer plugs; a pump connected to a reservoir that removably connects to the
tubes; a
transparent, optical grade glass layer attached to the bottom surface of the
second
micropatterned polymer layer that forms a bottom surface for the plurality of
wells and that
seals the multi-well plate perfusion culture device; wherein one or more of
the two adjacent
wells is a culture chamber for culturing a population of cells; (b) an
expanded population of
cells derived from a recipient subject comprising a cell-specific antigen in
the first adjacent
well of the device in (a); (c) an expanded population of T lymphocytes derived
from a
potential donor of a BMT graft in the second adjacent well of the device in
(a), wherein the
potential donor is allogeneic to the recipient subject; (d) a liquid culture
medium that is
flowable between the first adjacent well and the second adjacent well; the
model being
characterized by: circulation of the liquid medium from the first well into
the second well
and back to the first well through the microfluidic channel; an interaction
between the
population of cells comprising the cell antigen derived from the recipient
subject in the first
well and the population of T lymphocytes is effective to generate alloreactive
effector T
lymphocytes; alloreactive effector T lymphocyte-induced quantifiable damage to
the
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population of cells comprising the cell antigen derived from the recipient by
the population of
alloreactive effector T lymphocytes from the donor allogeneic to the
recipient.
[0025] According to one embodiment, the population of T lymphocytes in
(c) is
derived from peripheral blood lymphocytes of the potential donor. According to
another
embodiment, the population of T lymphocytes comprises a suspension of
nonadherent cells.
[0026] According to one embodiment, the first micropatterned polymer
layer and
the second micropatterned polymer layer comprises an organic polymer.
[0027] According to one embodiment, the organic polymer is
polydimethyl
siloxane (PMDS) or polystyrene.
[0028] According to one embodiment, the transparent polymer membrane
comprises a nanofibrous mesh. According to one embodiment, the nanofibrous
mesh is
placed on a top surface of the transparent polymer membrane to coat the top
surface of the
transparent polymer membrane. According to another embodiment, the nanofibrous
mesh
comprises a nanofibrous matrix comprising a plurality of pores through which
the population
of T lymphocytes derived from the potential donor allogeneic to the recipient
subject can
pass. According to another embodiment, the transparent polymer membrane
comprises a
plurality of microbeads preconditioned with an adhesion-promoting agent in an
amount
effective to promote adhesion of a population of cells to a surface of the
microbeads.
According to another embodiment, the adhesion promoting agent comprises a
lipopolysaccharide in an amount effective to promote adhesion of a
subpopulation of the
population of T lymphocytes to the microbead surface. According to another
embodiment,
the subpopulation of the population of T lymphocytes comprises a population of
dendritic
cells.
[0029] According to one embodiment, the suspension of nonadherent
cells
contains T lymphocytes derived from the allogeneic donor.
[0030] According to one embodiment, the population of alloreactive
effector T
lymphocytes comprises alloreactive activated antigen presenting cells.
According to another
embodiment, the alloreactive activated antigen presenting cells comprise a
population of
alloreactive activated dendritic cells.
[0031] According to one embodiment, the quantifiable damage to the
population
of cells comprising the cell-specific antigen derived from the recipient
induced by the
population of nonadherent alloreactive effector T lymphocytes from the donor
allogeneic to
the recipient comprises cell death.
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[0032] According to one embodiment, the population of cells comprising
the cell-
specific antigen in (b) are a population of intestinal epithelial cells
derived from the recipient
subject, and the alloreactive effector T lymphocyte-induced quantifiable
damage to the
population of intestinal epithelial cells of the recipient subject is an ex
vivo measure of risk of
graft vs. host disease.
[0033] According to another aspect, the described invention provides a
method
for optimizing donor selection for allogeneic blood and marrow transplantation
(BMT)
therapy comprising, in order: (a) acquiring a tissue sample from a recipient
subject
allogeneic to a potential donor of a BMT graft, the tissue sample comprising a
population of
primary intestinal epithelial cells comprising an intestinal epithelial cell-
specific antigen; (b)
seeding the population of primary intestinal epithelial cells of (a) in a
first adjacent well of a
multiwall plate-based perfusion culture device, the first adjacent well
comprising a
transparent polymer membrane, expanding the population in a first liquid
medium containing
ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2 fibroblast feeder layer
and
generating a population of conditional reprogrammed intestinal epithelial
cells (CRIECs)
comprising the intestinal cell-specific antigen derived from the recipient
subject; (c)
acquiring a population of T lymphocytes from the potential donor allogeneic to
the recipient;
(d) seeding and expanding in a second adjacent well of the multiwall plate-
based perfusion
culture device the population of T lymphocytes derived from the potential
donor of (c), (e)
co-culturing in a second liquid medium the CRIECs derived from the recipient
subject in the
first adjacent well and the T lymphocytes derived from the potential donor
allogeneic to the
recipient subject in the second adjacent well, the co-culturing being
characterized by: the
first adjacent well being fluidly connected to the second adjacent well so
that the second
liquid medium is flowable between the first adjacent well and the second
adjacent well; an
interaction between the population of CRIECs derived from the recipient
subject and the
population of T lymphocytes that is effective to generate alloreactive
effector T lymphocytes
derived from the potential allogeneic donor; (f) measuring damage to the
population of
CRIECs derived from the recipient subject induced by the alloreactive effector
T
lymphocytes derived from the potential donor allogeneic to the recipient
subject, wherein the
damage is a measure of a risk of intestinal graft versus host disease in the
recipient subject;
(g) ranking a plurality of potential donors by the measure of the risk of
intestinal graft versus
host disease; and (h) treating the recipient subject with a BMT graft derived
from a selected
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donor allogeneic to the recipient subject whose T lymphocytes are
characterized by a reduced
risk of intestinal graft-versus-host disease.
[0034] According to one embodiment, the potential donor of the BMT
graft is
haploidentical to the recipient subject.
[0035] According to one embodiment, the tissue sample comprising a
population
of primary intestinal epithelial cells is derived from small intestine, large
intestine or colon of
the recipient subject.
[0036] According to one embodiment, the transparent polymer membrane
comprises a nanofibrous mesh to which the population of CRIECs is adherent.
According to
another embodiment, the nanofibrous mesh comprises a nanofibrous matrix
comprising a
plurality of pores through which the population of T lymphocytes derived from
the potential
donor allogeneic to the recipient subject can pass. According to another
embodiment, the
transparent polymer membrane comprises a plurality of microbeads
preconditioned with an
amount of an adhesion-promoting agent effective to promote adhesion of a
population of
cells to at least one surface of the microbeads. According to another
embodiment, the
adhesion promoting agent comprises a lipopolysaccharide in an amount effective
to promote
adhesion of the population of cells.
[0037] According to one embodiment, the population of cells is a
subpopulation
of the population of T lymphocytes derived from the potential donor allogeneic
to the
recipient subject. According to another embodiment, the subpopulation of the
population of
T lymphocytes comprises a population of dendritic cells.
[0038] According to one embodiment, the expanded population of T
lymphocytes
derived from the donor allogeneic to the recipient subject in (d) comprise a
suspension of
nonadherent cells.
[0039] According to one embodiment, the population of alloreactive
effector T
lymphocytes comprises a population of alloreactive activated antigen
presenting cells.
According to another embodiment, the alloreactive activated antigen presenting
cells
comprise a population of alloreactive activated dendritic cells.
[0040] According to one embodiment, the quantifiable damage to the
population
of CRIECs derived from the recipient induced by the population of nonadherent
alloreactive
effector T lymphocytes from the donor allogeneic to the recipient comprises
cell death.
[0041] According to one embodiment, the method further comprising (i)
identifying T lymphocyte clones responsible for the quantifiable damage to the
population of
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CRIECs derived from the recipient subject; and (ii) selectively deleting the T
lymphocyte
clones from the population of T lymphocytes derived from the donor.
[0042]
According to another aspect, the described invention provides, a method
for minimizing risks and maximizing benefits of performing allogeneic blood
and marrow
transplantation (BMT) therapy in a recipient subject, wherein the recipient
subject has a
hematologic malignancy comprising, in order: (A) evaluating a population of T
lymphocytes
derived from a potential donor allogeneic to the recipient subject for a
potential to damage
intestinal epithelial cells of the recipient subject according to the method
of claim 17 steps (a)
through (g); (B) evaluating the population of T lymphocytes derived from the
potential donor
allogeneic to the recipient subject for an effective graft versus tumor
response against the
tumor-specific antigens by: (i) acquiring a specimen comprising a population
of tumor cells
derived from the recipient subject, the population of tumor cells comprising
one or more
tumor specific antigens; (ii) seeding and expanding the population of tumor
cells in the
second liquid medium in a third adjacent well of the multiwell plate-based
perfusion culture
device; the third adjacent well comprising a transparent polymer membrane;
(iii) acquiring a
population of T lymphocytes derived from the potential donor allogeneic to the
recipient
subject; (iv) seeding and expanding in a fourth adjacent well of the multiwall
plate-based
perfusion culture device the population of T lymphocytes of (iii), (v) co-
culturing in the
second liquid medium the population of tumor cells comprising one or more
tumor-specific
antigens that is derived from the recipient subject in the third adjacent well
and the
population of T lymphocytes derived from the potential donor allogeneic to the
recipient
subject in the fourth adjacent well, the co-culturing being characterized by:
the third adjacent
well being fluidly connected to the fourth adjacent well so that the second
liquid medium is
flowable between the third adjacent well and the fourth adjacent well; an
interaction between
the population of tumor cells comprising one or more tumor-specific antigens
that is derived
from the recipient subject and the population of T lymphocytes derived from
the potential
donor allogeneic to the recipient subject that is effective to generate
alloreactive effector T
lymphocytes derived from the potential donor; (vi) measuring damage to the
population of
tumor cells derived from the recipient subject induced by the alloreactive
effector T
lymphocytes derived from the potential donor allogeneic to the recipient
subject, wherein the
damage is a measure of an effective graft versus tumor (GVT) response against
the tumor-
specific antigens; (vii) ranking a plurality of potential donors by the
measure of the effective
GVT response against the tumor-specific antigens; and (C) treating the
recipient subject with
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a BMT graft derived from a selected donor allogeneic to the recipient, the
selected donor
being characterized by a reduced risk of intestinal graft versus host disease,
and an effective
GVT response against the tumor-specific antigens.
[0043] According to one embodiment, the potential donor of the BMT
graft is
haploidentical to the recipient subject.
[0044] According to one embodiment, the specimen comprising the
population of
tumor cells derived from the recipient subject in (i) is a blood sample, a
bone marrow sample,
or a leukapheresis sample.
[0045] According to one embodiment, the transparent polymer membrane
comprises a nanofibrous mesh to which the population of tumor cells is
adherent. According
to another embodiment, the nanofibrous mesh comprises a nanofibrous matrix
comprising a
plurality of pores through which the population of T lymphocytes derived from
the potential
donor allogeneic to the recipient subject can pass. According to another
embodiment, the
transparent polymer membrane comprises a plurality of microbeads
preconditioned with an
amount of an adhesion-promoting agent effective to promote adhesion of a
population of
cells to at least one surface of the microbeads. According to another
embodiment, the
adhesion promoting agent comprises a lipopolysaccharide in an amount effective
to promote
adhesion of the population of cells.
[0046] According to one embodiment, the population of cells is a
subpopulation
of the population of T lymphocytes derived from the potential donor allogeneic
to the
recipient subject. According to another embodiment, the subpopulation of the
population of
T lymphocytes comprises a population of dendritic cells.
[0047] According to one embodiment, the expanded population of T
lymphocytes
derived from the allogeneic donor in (iv) comprise a suspension of nonadherent
cells.
[0048] According to another embodiment, the population of alloreactive
effector
T lymphocytes in (v) comprises a population of alloreactive activated antigen
presenting
cells. According to another embodiment, the alloreactive activated antigen
presenting cells
comprise a population of alloreactive activated dendritic cells.
[0049] According to one embodiment, the quantifiable damage to the
population
of tumor cells derived from the recipient subject induced by the population of
nonadherent
alloreactive effector T lymphocytes derived from the potential donor
allogeneic to the
recipient comprises cell death.
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[0050] According to one embodiment, the method further comprising
enriching
the population of T lymphocytes derived from the potential donor for an
effective GVT
therapeutic effect by (i) identifying T lymphocyte clones responsible for the
quantifiable
damage to the population of tumor cells derived from the recipient subject;
and (ii) selecting
the T lymphocyte clones from the population of T lymphocytes derived from the
potential
donor allogeneic to the recipient subject; and (iii) expanding the T
lymphocyte clones to
obtain a therapeutic amount of the T cell clones effective to mediate a GVT
response against
the tumor-specific antigens.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] For a more complete understanding of the described invention,
reference is
made to the following detailed description of an exemplary embodiment
considered in
conjunction with the accompanying drawings, in which:
[0052] FIG. 1 shows a drawing of an embodiment of the described
multiwell
plate-based perfusion culture device for co-cultivating recipient subject
intestinal epithelial
cells and an allogeneic donor's T lymphocytes.
[0053] FIG. 2(a) and 2(b) show immunofluorescence analysis of m-
CRIECs.
Staining was performed using a directly conjugated anti-pan cytokertin
antibody on mCRIEC
cells. 50 px scale-bar equals 16.1 p.m (20x) and 8.05 p.m (40x). Figure 2(c)
shows flow
cytometric analysis of extracellular (pan cytokeratin, EpCAM, CD24, and CD44)
and
intracellular (Lgr5) IEC cell markers.
[0054] FIG. 3 shows upregulation of MHC I and II in m-CRIEC. M-CRIEC
were
cultured in different cell culture media for 72 h in the presence of 20 ng/ml
TNF-a and 10
U/ml IFN-y to induce the upregulation of MHC class 1 (lab) and class II (H2kb)
molecules on
the surface of m-CRIEC. Maximal MHC II expression was obtained when using
cRPMI
medium or cRPMI plus a nanofibrous mesh.
[0055] FIG. 4 shows morphology of m-CRIECs cultured in CRC medium,
cRPMI
and cRPMI on nanofibrous mesh for 7 days to evaluate morphology and viability.
Loss of
cobble stone morphology (black arrow) was observed without nanofibrous mesh.
[0056] FIG. 5 shows a killing assay of B10.BR T cells against B6 m-
CRIEC. B6
m-CRIEC cultured with nanofibrous mesh and cRPMI were stimulated with 20 ng/ml
TNF-a
and 10 U/ml IFN-y for 48-72 h (panel I) and cocultured with MLC-stimulated
B10.BR T cells
at an E:T ratio of 5:1 (panel II) and 10:1 (panel III). On day 6, m-CRIEC at
E:T ratio 10:1
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were visibly compromised as determined by trypan blue staining (panel III).
Arrows indicate
T cells.
[0057] FIG. 6 shows a well plate-based perfusion device: (a) ¨ (c)
schematic
illustrations of the device and fabrication; (d) actual device used for
preliminary results; (e)
SEM image of nanofibrous mesh coated onto a PC membrane.
[0058] FIG. 7 (a) m-CRIEC from B6 mice were cultured for 7 days in the
IEC
culture chamber; (b) and (c) followed by the introduction of circulating MHC-
mismatched
B10.BR m-T cells for 5 more days. (b) SEM and (c) fluorescence images showing
m-
CRIECs and m-T cells. Scale bar: 50 p.m.
[0059] FIG. 8 shows flow cytometric analysis of T cell proliferation
upon DC
stimulation in 2D vs. 3D culture. eGFP B6 T cells were labeled with eFluor 670
and cultured
for 4 days with BALB.B DCs to assess proliferation. In comparison to 2D, the
total percent
(%) of live T cells [undivided cells (U) + proliferating (P)] was greater in
3D, indicating more
T cells remained viable in 3D perfusion culture. This enhanced viability was
also reflected
by the increased R:S ratio (10:1 vs. 2:1). Furthermore, the percentage of
proliferating T cells
(P) was greater in 3D than in 2D (40% vs. 33%).
[0060] FIG. 9 shows circulation of primary murine T cells through
primary
murine IECs: (a) schematic illustration of the device configuration and use;
(b) SEM image
showing collagen/PCL nanofiber mesh on PC membrane with 101.tm pores; (c)
merged
fluorescence image showing IEC cytoskeleton (red, ActinRed) and nucleus (blue,
DAPI)
after day 7 on collagen/PCL nanofiber mesh; (d) effect of IEC presence on T
cell viability;
and (e) bright field image showing IECs and T cells cultured for 6 h on
nanofiber mesh.
DETAILED DESRIPTION OF THE INVENTION
Glossary
[0061] Various terms used throughout this specification shall have the
definitions
set out herein.
[0062] The term "activation" or "lymphocyte activation" refers to
stimulation of
lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells
resulting in
synthesis of RNA, protein and DNA and production of lymphokines; it is
followed by
proliferation and differentiation of various effector and memory cells. For
example, a mature
B cell can be activated by an encounter with an antigen that expresses
epitopes that are
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recognized by its cell surface immunoglobulin (Ig). The activation process may
be a direct
one, dependent on cross-linkage of membrane Ig molecules by the antigen (cross-
linkage-
dependent B cell activation) or an indirect one, occurring most efficiently in
the context of an
intimate interaction with a helper T cell ("cognate help process"). T-cell
activation is
dependent on the interaction of the TCR/CD3 complex with its cognate ligand, a
peptide
bound in the groove of a class I or class II MHC molecule. The molecular
events set in
motion by receptor engagement are complex. Among the earliest steps appears to
be the
activation of tyrosine kinases leading to the tyrosine phosphorylation of a
set of substrates
that control several signaling pathways. These include a set of adapter
proteins that link the
TCR to the ras pathway, phospholipase Cyl, the tyrosine phosphorylation of
which increases
its catalytic activity and engages the inositol phospholipid metabolic
pathway, leading to
elevation of intracellular free calcium concentration and activation of
protein kinase C, and a
series of other enzymes that control cellular growth and differentiation. Full
responsiveness
of a T cell requires, in addition to receptor engagement, an accessory cell-
delivered
costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and/or
CD86 on the
antigen presenting cell (APC). The soluble product of an activated B
lymphocyte is
immmunoglobulins (antibodies). The soluble product of an activated T
lymphocyte is
lymphokines.
[0063] The term "administering" as used herein includes in vivo
administration,
as well as administration directly to tissue ex vivo. Generally, compositions
can be
administered systemically either orally, buccally, parenterally, topically, by
inhalation or
insufflation (i.e., through the mouth or through the nose), or rectally in
dosage unit
formulations containing conventional nontoxic pharmaceutically acceptable
carriers,
adjuvants, and vehicles as desired, or can be locally administered by means
such as, but not
limited to, injection, implantation, grafting, topical application, or
parenterally.
[0064] The term "alloantigen" as used herein refers to a genetically
determined
antigen present in some but not all individuals of a species (as those of a
particular blood
group) and capable of inducing the production of an alloantibody by
individuals which lack
it¨called also isoantigen.
[0065] The term "allogeneic" as used herein refers to taken from
different
individuals of the same species.
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[0066] The term "allogeneic bone marrow transplantation"(allo-BMT) as
used
herein refers to a procedure in which a recipient subject receives cells from
a genetically
similar, but not identical, donor.
[0067] The term "alloreactivity" refers to a strong primary T cell
response against
allelic variants of major histocompatibility complex (MHC) molecules in a
species.
Alloreactivity is manifested in the rejection of tissue grafts between
individuals of the same
species.
[0068] The term "antigen presenting cell" or APC as used herein refers
to a cell
that displays foreign antigen complexed with MHC molecules on its surface.
[0069] The terms "apoptosis" or "programmed cell death" refer to a
highly
regulated and active process that contributes to biologic homeostasis
comprised of a series of
biochemical events that lead to a variety of morphological changes, including
blebbing,
changes to the cell membrane, such as loss of membrane asymmetry and
attachment, cell
shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA
fragmentation, without damaging the organism.
[0070] Apoptotic cell death is induced by many different factors and
involves
numerous signaling pathways, some dependent on caspase proteases (a class of
cysteine
proteases) and others that are caspase independent. It can be triggered by
many different
cellular stimuli, including cell surface receptors, mitochondrial response to
stress, and
cytotoxic T cells, resulting in activation of apoptotic signaling pathways
[0071] The caspases involved in apoptosis convey the apoptotic signal
in a
proteolytic cascade, with caspases cleaving and activating other caspases that
then degrade
other cellular targets that lead to cell death. The caspases at the upper end
of the cascade
include caspase-8 and caspase-9. Caspase-8 is the initial caspase involved in
response to
receptors with a death domain (DD) like Fas.
[0072] Receptors in the TNF receptor family are associated with the
induction of
apoptosis, as well as inflammatory signaling. The Fas receptor (CD95) mediates
apoptotic
signaling by Fas-ligand expressed on the surface of other cells. The Fas-FasL
interaction
plays an important role in the immune system and lack of this system leads to
autoimmunity,
indicating that Fas-mediated apoptosis removes self-reactive lymphocytes. Fas
signaling also
is involved in immune surveillance to remove transformed cells and virus
infected cells.
Binding of Fas to oligimerized FasL on another cell activates apoptotic
signaling through a
cytoplasmic domain termed the death domain (DD) that interacts with signaling
adaptors
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including FAF, FADD and DAX to activate the caspase proteolytic cascade.
Caspase-8 and
caspase-10 first are activated to then cleave and activate downstream caspases
and a variety
of cellular substrates that lead to cell death.
[0073] Mitochondria participate in apoptotic signaling pathways
through the
release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key
protein in electron
transport, is released from mitochondria in response to apoptotic signals, and
activates Apaf-
1, a protease released from mitochondria. Activated Apaf-1 activates caspase-9
and the rest
of the caspase pathway. Smac/DIABLO is released from mitochondria and inhibits
IAP
proteins that normally interact with caspase-9 to inhibit apoptosis. Apoptosis
regulation by
Bc1-2 family proteins occurs as family members form complexes that enter the
mitochondrial
membrane, regulating the release of cytochrome c and other proteins. TNF
family receptors
that cause apoptosis directly activate the caspase cascade, but can also
activate Bid, a Bc1-2
family member, which activates mitochondria-mediated apoptosis. Bax, another
Bc1-2 family
member, is activated by this pathway to localize to the mitochondrial membrane
and increase
its permeability, releasing cytochrome c and other mitochondrial proteins. Bc1-
2 and Bc1-xL
prevent pore formation, blocking apoptosis. Like cytochrome c, AIF (apoptosis-
inducing
factor) is a protein found in mitochondria that is released from mitochondria
by apoptotic
stimuli. While cytochrome C is linked to caspase-dependent apoptotic
signaling, AIF release
stimulates caspase-independent apoptosis, moving into the nucleus where it
binds DNA.
DNA binding by AIF stimulates chromatin condensation, and DNA fragmentation,
perhaps
through recruitment of nucleases.
[0074] The mitochondrial stress pathway begins with the release of
cytochrome c
from mitochondria, which then interacts with Apaf-1, causing self-cleavage and
activation of
caspase-9. Caspase-3, -6 and-7 are downstream caspases that are activated by
the upstream
proteases and act themselves to cleave cellular targets.
[0075] Granzyme B and perforin proteins released by cytotoxic T cells
induce
apoptosis in target cells, forming transmembrane pores, and triggering
apoptosis, perhaps
through cleavage of caspases, although caspase-independent mechanisms of
Granzyme B
mediated apoptosis have been suggested.
[0076] Fragmentation of the nuclear genome by multiple nucleases
activated by
apoptotic signaling pathways to create a nucleosomal ladder is a cellular
response
characteristic of apoptosis. One nuclease involved in apoptosis is DNA
fragmentation factor
(DFF), a caspase-activated DNAse (CAD). DFF/CAD is activated through cleavage
of its
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associated inhibitor ICAD by caspases proteases during apoptosis. DFF/CAD
interacts with
chromatin components such as topoisomerase II and histone H1 to condense
chromatin
structure and perhaps recruit CAD to chromatin. Another apoptosis activated
protease is
endonuclease G (EndoG). EndoG is encoded in the nuclear genome but is
localized to
mitochondria in normal cells. EndoG may play a role in the replication of the
mitochondrial
genome, as well as in apoptosis. Apoptotic signaling causes the release of
EndoG from
mitochondria. The EndoG and DFF/CAD pathways are independent since the EndoG
pathway still occurs in cells lacking DFF.
[0077] Hypoxia, as well as hypoxia followed by reoxygenation can
trigger
cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) a serine-
threonine
kinase ubiquitously expressed in most cell types, appears to mediate or
potentiate apoptosis
due to many stimuli that activate the mitochondrial cell death pathway.
Loberg, RD, et al., J.
Biol. Chem. 277 (44): 41667-673 (2002). It has been demonstrated to induce
caspase 3
activation and to activate the proapoptotic tumor suppressor gene p53. It also
has been
suggested that GSK-3 promotes activation and translocation of the proapoptotic
Bc1-2 family
member, Bax, which, upon agregation and mitochondrial localization, induces
cytochrome c
release. Akt is a critical regulator of GSK-3, and phosphorylation and
inactivation of GSK-3
may mediate some of the antiapoptotic effects of Akt.
[0078] The term "chemokine" as used herein refers to a class of
chemotactic
cytokines that signal leukocytes to move in a specific direction. The terms
"chemotaxis" or
"chemotactic" refer to the directed motion of a motile cell or part along a
chemical
concentration gradient towards environmental conditions it deems attractive
and/or away
from surroundings it finds repellent.
[0079] The term "conditioning regimens" as used herein refers to
treatments used
to prepare a patient for stem cell transplantation (a procedure in which a
person receives
blood stem cells, which make any type of blood cell). A conditioning regimen
may include
chemotherapy, monoclonal antibody therapy, and radiation to the entire body.
It helps make
room in the patient's bone marrow for new blood stem cells to grow, helps
prevent the
patient's body from rejecting the transplanted cells, and helps kill any
cancer cells that are in
the body.
[0080] The term "conditional reprogramming cells (CRC)" as used herein
refers
to epithelial cells cultured on irradiated fibroblast feeders in the presence
of the Rho kinase
inhibitor Y-27632. Saenz, F.R. et al., "Conditionally reprogrammed normal and
transformed
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mouse mammary epithelial cells display a progenitor-cell-like phenotype," PloS
One 9,
e9766, doi: 10.1371/journal.pone.0097666 (2014); Palechor-Ceron, N. et al,
"Radiation
induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to
conditionally
reprogram and immortalize epithelial cells," Am. J. Pathol. 183: 1862-70, doi:
10.1016/j.ajpath.2013.08.009 (2013); Liu, X. et al, "ROCK inhibitor and feeder
cells induce
the conditional reprogramming of epithelial cells," Am. J. Pathol. 180: 599-
607, doi:
10.1016/j.ajpath.2011.10.036 (2012). The inductive conditions consist of F
medium
containing the ROCK inhibitor Y-27632 and irradiated Swiss 3T3-J2 mouse
fibroblasts.
When the feeder cells and Y-27632 were removed, the CRCs exhibited normal
differentiation. The CRCs are genetically stable, can be cultured
indefinitely, and can bypass
senescence.
[0081] The term "condition", as used herein, refers to a variety of
health states
and is meant to include disorders or diseases caused by any underlying
mechanism or injury.
[0082] The term "culture" as used herein refers to the cultivation of
cells in or on
a controlled or defined medium. The terms "culture-expanded" or "expanded" are
used
interchangeably to refer to an increase in the number of cells by cultivation
of the cells in or
on a controlled or defined medium.
[0083] The term "cytokine" as used herein refers to small soluble
protein
substances secreted by cells, which have a variety of effects on other cells.
Cytokines
mediate many important physiological functions, including growth, development,
wound
healing, and the immune response. They act by binding to their cell-specific
receptors located
in the cell membrane, which allows a distinct signal transduction cascade to
start in the cell,
which eventually will lead to biochemical and phenotypic changes in target
cells. Generally,
cytokines act locally. They include type I cytokines, which encompass many of
the
interleukins including interleukin 2 (IL-2), as well as several hematopoietic
growth factors;
type II cytokines, including the interferons and interleukin-10; tumor
necrosis factor
("TNF")-related molecules, including TNFa and lymphotoxin; immunoglobulin
super-family
members, including interleukin 1 ("ILA"); and the chemokines, a family of
molecules that
play a critical role in a wide variety of immune and inflammatory functions.
The same
cytokine can have different effects on a cell depending on the state of the
cell. Cytokines
often regulate the expression of, and trigger cascades of, other cytokines.
[0084] The term "dendritic cells" (DCs) as used herein, refers to
professional
APCs capable of presenting both MHC-I and MHC-II antigens.
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[0085] The term "derived from" as used herein is used to refer to
originating,
sourced, or coming from.
[0086] The term "disease" or "disorder," as used herein, refers to an
impairment
of health or a condition of abnormal functioning.
[0087] The term "drug" as used herein refers to a therapeutic agent or
any
substance used in the prevention, diagnosis, alleviation, treatment, or cure
of disease.
[0088] The term "graft" as used herein, refers to any tissue or organ
for
transplantation. It includes, but is not limited to, a self-tissue transferred
from one body site to
another in the same individual ("autologous graft"), a tissue transferred
between genetically
identical individuals or sufficiently immunologically compatible to allow
tissue transplant
("syngeneic graft"), a tissue transferred between genetically different
members of the same
species ("allogeneic graft" or "allograft"), and a tissue transferred between
different species
("xenograft").
[0089] The term "graft versus host" as used herein, refers to a
systemic
autoimmune syndrome resulting from cells of an engrafted hematopoietic-cell
transplant
mounting an immune response against the host. In human recipients of bone
marrow, chronic
GVHD syndrome is a major clinical problem, leading to fibrosis, pathology and
autoantibodies, which can result in immune dysfunction, increased risk of
infection,
potentially serious impaired organ function, and poor quality of life. The
syndrome occurs
even in recipients of autologous marrow, although in a milder form. (See, e.g.
Kennedy,
Autologous graft versus host disease. Med. Oncol. 12: 149-15;1995). Acute GVHD
is a
clinical syndrome caused by T cell-mediated recognition of minor
histocompatibility antigens
followed by organ-specific vascular adhesion, migration, proliferation,
cytokine release, and
direct cell-mediated attack on normal tissues. Chronic GVHD is more complex,
incorporating both conventional T-cell effector functions, as well as humoral
and antigen-
presenting effects of B cells. (Joseph Antin, "T-cell depletion in GVHD: less
is more?" Blood
(2011) 117(23): 6061-6063)
[0090] The term "graft-versus-tumor (GVT)" as used herein refers to an
immune
response to a graft recipient's tumor cells by immune cells present in a
donor's transplanted
tissue, such as bone marrow or peripheral blood.
[0091] The term "haploidentical" as used herein refers to sharing a
haplotype,
meaning having the same alleles at a set of closely linked genes on one
chromosome.
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[0092] The term "human leukocyte antigen matching" as used herein
refers to a
process in which blood or tissue samples are tested for human leukocyte
antigens (HLAs).
HLAs are molecules found on the surface of most cells in the body. They make
up a person's
tissue type, which varies from person to person. They play an important part
in the body's
immune response to foreign substances. Human leukocyte antigen matching is
done before a
donor stem cell or organ transplant to find out if tissues match between the
donor and the
person receiving the transplant. Also called HLA matching.
[0093] The term "hematopoietic-cell transplantation" (HCT) is used
herein to
refer to blood and marrow transplantation (BMT), a procedure that involves
infusion of cells
(hematopoietic stem cells; also called hematopoietic progenitor cells) to
reconstitute the
hematopoietic system of a patient.
[0094] The term "inhibit" and its various grammatical forms,
including, but not
limited to, "inhibiting" or "inhibition", are used herein to refer to reducing
the amount or rate
of a process, to stopping the process entirely, or to decreasing, limiting, or
blocking the action
or function thereof. Inhibition can include a reduction or decrease of the
amount, rate, action
function, or process of a substance by at least 5%, at least 10%, at least
15%, at least 20%, at
least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least
55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at
least 98%, or at least 99%.
[0095] The term "inhibitor" as used herein refers to a second molecule
that binds
to a first molecule thereby decreasing the first molecule's activity. Enzyme
inhibitors are
molecules that bind to enzymes thereby decreasing enzyme activity. The binding
of an
inhibitor can stop a substrate from entering the active site of the enzyme
and/or hinder the
enzyme from catalyzing its reaction. Inhibitor binding is either reversible or
irreversible.
Irreversible inhibitors usually react with the enzyme and change it
chemically, for example,
by modifying key amino acid residues needed for enzymatic activity. In
contrast, reversible
inhibitors bind non-covalently and produce different types of inhibition
depending on
whether these inhibitors bind the enzyme, the enzyme-substrate complex, or
both. Enzyme
inhibitors often are evaluated by their specificity and potency.
[0096] The term "injury," as used herein, refers to damage or harm to
a structure
or function of the body caused by an outside agent or force, which can be
physical or
chemical.
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[0097] The term "immunomodulatory cell(s)" as used herein refer(s) to
cell(s) that
are capable of augmenting or diminishing immune responses by expressing
chemokines,
cytokines and other mediators of immune responses.
[0098] The term "inflammatory cytokines" or "inflammatory mediators"
as used
herein refers to the molecular mediators of the inflammatory process, which
may modulate
being either pro- or anti-inflamatory in their effect. These soluble,
diffusible molecules act
both locally at the site of tissue damage and infection and at more distant
sites. Some
inflammatory mediators are activated by the inflammatory process, while others
are
synthesized and/or released from cellular sources in response to acute
inflammation or by
other soluble inflammatory mediators. Examples of inflammatory mediators of
the
inflammatory response include, but are not limited to, plasma proteases,
complement, kinins,
clotting and fibrinolytic proteins, lipid mediators, prostaglandins,
leukotrienes, platelet-
activating factor (PAF), peptides and amines, including, but not limited to,
histamine,
serotonin, and neuropeptides, pro-inflammatory cytokines, including, but not
limited to,
interleukin-l-beta (IL-1(3), interleukin-4 (IL-4), interleukin-6 (IL-6),
interleukin-8 (IL-8),
tumor necrosis factor-alpha (TNF-a), interferon-gamma (IF-y), and interleukin-
12 (IL-12).
[0099] The term "interleukin (IL)" as used herein refers to a cytokine
secreted by,
and acting on, leukocytes. Interleukins regulate cell growth, differentiation,
and motility, and
stimulates immune responses, such as inflammation. Examples of interleukins
include
interleukin-1 (IL-1), interleukin 2 (IL-2), interleukin-1(3 (IL-1(3),
interleukin-6 (IL-6),
interleukin-8 (IL-8), and interleukin-12 (IL-12).
[00100] The term "lymphocyte" refers to a small white blood cell formed in
lymphatic tissue throughout the body and in normal adults making up about 22-
28% of the
total number of leukocytes in the circulating blood that plays a large role in
defending the
body against disease. Individual lymphocytes are specialized in that they are
committed to
respond to a limited set of structurally related antigens. This commitment,
which exists
before the first contact of the immune system with a given antigen, is
expressed by the
presence on the lymphocyte's surface membrane of receptors specific for
determinants
(epitopes) on the antigen. Each lymphocyte possesses a population of
receptors, all of which
have identical combining sites. One set, or clone, of lymphocytes differs from
another clone
in the structure of the combining region of its receptors and thus differs in
the epitopes that it
can recognize. Lymphocytes differ from each other not only in the specificity
of their
receptors, but also in their functions.
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[00101] Two broad classes of lymphocytes are recognized: the B-lymphocytes (B-
cells), which are precursors of antibody-secreting cells, and T-lymphocytes (T-
cells),
B-lymphocytes
[00102] B-lymphocytes are derived from hematopoietic cells of the bone marrow.
A mature B-cell can be activated with an antigen that expresses epitopes that
are recognized
by its cell surface. The activation process may be direct, dependent on cross-
linkage of
membrane Ig molecules by the antigen (cross-linkage-dependent B-cell
activation), or
indirect, via interaction with a helper T-cell, in a process referred to as
cognate help. In many
physiological situations, receptor cross-linkage stimuli and cognate help
synergize to yield
more vigorous B-cell responses. (Paul, W. E., "Chapter 1: The immune system:
an
introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-
Raven
Publishers, Philadelphia (1999)).
[00103] Cross-linkage dependent B-cell activation requires that the antigen
express
multiple copies of the epitope complementary to the binding site of the cell
surface receptors
because each B-cell expresses Ig molecules with identical variable regions.
Such a
requirement is fulfilled by other antigens with repetitive epitopes, such as
capsular
polysaccharides of microorganisms or viral envelope proteins. Cross-linkage-
dependent B-
cell activation is a major protective immune response mounted against these
microbes. (Paul,
W. E., "Chapter 1: The immune system: an introduction," Fundamental
Immunology, 4th
Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
[00104] Cognate help allows B-cells to mount responses against antigens that
cannot cross-link receptors and, at the same time, provides costimulatory
signals that rescue
B cells from inactivation when they are stimulated by weak cross-linkage
events. Cognate
help is dependent on the binding of antigen by the B-cell's membrane
immunoglobulin (Ig),
the endocytosis of the antigen, and its fragmentation into peptides within the
endosomal/lysosomal compartment of the cell. Some of the resultant peptides
are loaded into
a groove in a specialized set of cell surface proteins known as class II major
histocompatibility complex (MHC) molecules. The resultant class II/peptide
complexes are
expressed on the cell surface and act as ligands for the antigen-specific
receptors of a set of
T-cells designated as CD4+ T-cells. The CD4+ T-cells bear receptors on their
surface
specific for the B-cell's class II/peptide complex. B-cell activation depends
not only on the
binding of the T cell through its T cell receptor (TCR), but this interaction
also allows an
activation ligand on the T-cell (CD40 ligand) to bind to its receptor on the B-
cell (CD40)
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signaling B-cell activation. In addition, T helper cells secrete several
cytokines that regulate
the growth and differentiation of the stimulated B-cell by binding to cytokine
receptors on the
B cell. (Paul, W. E., "Chapter 1: The immune system: an introduction,"
Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,
Philadelphia (1999)).
[00105] During cognate help for antibody production, the CD40 ligand is
transiently expressed on activated CD4+ T helper cells, and it binds to CD40
on the antigen-
specific B cells, thereby tranducing a second costimulatory signal. The latter
signal is
essential for B cell growth and differentiation and for the generation of
memory B cells by
preventing apoptosis of germinal center B cells that have encountered antigen.
Hyperexpression of the CD40 ligand in both B and T cells is implicated in the
pathogenic
autoantibody production in human SLE patients. (Desai-Mehta, A. et al.,
"Hyperexpression
of CD40 ligand by B and T cells in human lupus and its role in pathogenic
autoantibody
production," J. Clin. Invest. 97(9): 2063-2073 (1996)).
T-lymphocytes
[00106] T-lymphocytes derive from precursors in hematopoietic tissue, undergo
differentiation in the thymus, and are then seeded to peripheral lymphoid
tissue and to the
recirculating pool of lymphocytes. T-lymphocytes or T cells mediate a wide
range of
immunologic functions. These include the capacity to help B cells develop into
antibody-
producing cells, the capacity to increase the microbicidal action of
monocytes/macrophages,
the inhibition of certain types of immune responses, direct killing of target
cells, and
mobilization of the inflammatory response. These effects depend on their
expression of
specific cell surface molecules and the secretion of cytokines. (Paul, W. E.,
"Chapter 1: The
immune system: an introduction," Fundamental Immunology, 4th Edition, Ed.
Paul, W. E.,
Lippicott-Raven Publishers, Philadelphia (1999)).
[00107] T cells differ from B cells in their mechanism of antigen recognition.
Immunoglobulin, the B cell's receptor, binds to individual epitopes on soluble
molecules or
on particulate surfaces. B-cell receptors see epitopes expressed on the
surface of native
molecules. Antibody and B-cell receptors evolved to bind to and to protect
against
microorganisms in extracellular fluids. In contrast, T cells recognize
antigens on the surface
of other cells and mediate their functions by interacting with, and altering,
the behavior of
these antigen-presenting cells (APCs). There are three main types of antigen-
presenting cells
in peripheral lymphoid organs that can activate T cells: dendritic cells,
macrophages and B
cells. The most potent of these are the dendritic cells, whose only function
is to present
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foreign antigens to T cells. Immature dendritic cells are located in tissues
throughout the
body, including the skin, gut, and respiratory tract. When they encounter
invading microbes
at these sites, they endocytose the pathogens and their products, and carry
them via the lymph
to local lymph nodes or gut associated lymphoid organs. The encounter with a
pathogen
induces the dendritic cell to mature from an antigen-capturing cell to an
antigen-presenting
cell (APC) that can activate T cells. APCs display three types of protein
molecules on their
surface that have a role in activating a T cell to become an effector cell:
(1) MHC proteins,
which present foreign antigen to the T cell receptor; (2) costimulatory
proteins which bind to
complementary receptors on the T cell surface; and (3) cell-cell adhesion
molecules, which
enable a T cell to bind to the antigen-presenting cell (APC) for long enough
to become
activated. ("Chapter 24: The adaptive immune system," Molecular Biology of the
Cell,
Alberts, B. et al., Garland Science, NY, 2002).
[00108] T-cells are subdivided into two distinct classes based on the
cell surface
receptors they express. The majority of T cells express T cell receptors (TCR)
consisting of a
and 0 chains. A small group of T cells express receptors made of y and 6
chains. Among the
a/f3 T cells are two important sublineages: those that express the coreceptor
molecule CD4
(CD4+ T cells); and those that express CD8 (CD8+ T cells). These cells differ
in how they
recognize antigen and in their effector and regulatory functions.
[00109] CD4+ T cells are the major regulatory cells of the immune system.
Their
regulatory function depends both on the expression of their cell-surface
molecules, such as
CD40 ligand whose expression is induced when the T cells are activated, and
the wide array
of cytokines they secrete when activated.
[00110] T cells also mediate important effector functions, some of which are
determined by the patterns of cytokines they secrete. The cytokines can be
directly toxic to
target cells and can mobilize potent inflammatory mechanisms.
[00111] In addition, T cells particularly CD8+ T cells, can develop into
cytotoxic
T-lymphocytes (CTLs) capable of efficiently lysing target cells that express
antigens
recognized by the CTLs. (Paul, W. E., "Chapter 1: The immune system: an
introduction,"
Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven
Publishers,
Philadelphia (1999)).
[00112] T cell receptors (TCRs) recognize a complex consisting of a peptide
derived by proteolysis of the antigen bound to a specialized groove of a class
II or class I
MHC protein. The CD4+ T cells recognize only peptide/class II complexes while
the CD8+
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T cells recognize peptide/class I complexes. (Paul, W. E., "Chapter 1: The
immune system:
an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,
Lippicott-Raven
Publishers, Philadelphia (1999)).
[00113] The TCR's ligand (i.e., the peptide/MHC protein complex) is created
within antigen-presenting cells (APCs). In general, class II MHC molecules
bind peptides
derived from proteins that have been taken up by the APC through an endocytic
process.
These peptide-loaded class II molecules are then expressed on the surface of
the cell, where
they are available to be bound by CD4+ T cells with TCRs capable of
recognizing the
expressed cell surface complex. Thus, CD4+ T cells are specialized to react
with antigens
derived from extracellular sources. (Paul, W. E., "Chapter 1: The immune
system: an
introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-
Raven
Publishers, Philadelphia (1999)).
[00114] In contrast, class I MHC molecules are mainly loaded with peptides
derived from internally synthesized proteins, such as viral proteins. These
peptides are
produced from cytosolic proteins by proteolysis by the proteosome and are
translocated into
the rough endoplasmic reticulum. Such peptides, generally nine amino acids in
length, are
bound into the class I MHC molecules and are brought to the cell surface,
where they can be
recognized by CD8+ T cells expressing appropriate receptors. This gives the T
cell system,
particularly CD8+ T cells, the ability to detect cells expressing proteins
that are different
from, or produced in much larger amounts than, those of cells of the remainder
of the
organism (e.g., vial antigens) or mutant antigens (such as active oncogene
products), even if
these proteins in their intact form are neither expressed on the cell surface
nor secreted.
(Paul, W. E., "Chapter 1: The immune system: an introduction," Fundamental
Immunology,
4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia
(1999)).
[00115] T cells can also be classified based on their function as
helper T cells; T
cells involved in inducing cellular immunity; suppressor T cells; and
cytotoxic T cells.
Helper T cells
[00116] Helper T cells are T cells that stimulate B cells to make antibody
responses
to proteins and other T cell-dependent antigens. T cell-dependent antigens are
immunogens
in which individual epitopes appear only once or a limited number of times
such that they are
unable to cross-link the membrane immunoglobulin (Ig) of B cells or do so
inefficiently. B
cells bind the antigen through their membrane Ig, and the complex undergoes
endocytosis.
Within the endosomal and lysosomal compartments, the antigen is fragmented
into peptides
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by proteolytic enzymes and one or more of the generated peptides are loaded
into class II
MHC molecules, which traffic through this vesicular compartment. The resulting
peptide/class II MHC complex is then exported to the B-cell surface membrane.
T cells with
receptors specific for the peptide/class II molecular complex recognize this
complex on the
B-cell surface. (Paul, W. E., "Chapter 1: The immune system: an introduction,"
Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,
Philadelphia (1999)).
[00117] B-cell activation depends both on the binding of the T cell through
its TCR
and on the interaction of the T-cell CD40 ligand (CD4OL) with CD40 on the B
cell. T cells
do not constitutively express CD4OL. Rather, CD4OL expression is induced as a
result of an
interaction with an APC that expresses both a cognate antigen recognized by
the TCR of the
T cell and CD80 or CD86. CD80/CD86 is generally expressed by activated, but
not resting,
B cells so that the helper interaction involving an activated B cell and a T
cell can lead to
efficient antibody production. In many cases, however, the initial induction
of CD4OL on T
cells is dependent on their recognition of antigen on the surface of APCs that
constitutively
express CD80/86, such as dendritic cells. Such activated helper T cells can
then efficiently
interact with and help B cells. Cross-linkage of membrane Ig on the B cell,
even if
inefficient, may synergize with the CD4OL/CD40 interaction to yield vigorous B-
cell
activation. The subsequent events in the B-cell response, including
proliferation, Ig
secretion, and class switching (of the Ig class being expressed) either depend
or are enhanced
by the actions of T cell-derived cytokines. (Paul, W. E., "Chapter 1: The
immune system:
an introduction," Fundamental Immunology, 4th Edition, Ed. Paul, W. E.,
Lippicott-Raven
Publishers, Philadelphia (1999)).
[00118] CD4+ T cells tend to differentiate into cells that principally
secrete the
cytokines IL-4, IL-5, IL-6, and IL-10 (TH2 cells) or into cells that mainly
produce IL-2, IFN-
0 , and lymphotoxin (TH1 cells). The TH2 cells are very effective in helping B-
cells develop
into antibody-producing cells, whereas the TH1 cells are effective inducers of
cellular
immune responses, involving enhancement of microbicidal activity of monocytes
and
macrophages, and consequent increased efficiency in lysing microorganisms in
intracellular
vesicular compartments. Although the CD4+ T cells with the phenotype of TH2
cells (i.e.,
IL-4, IL-5, IL-6 and IL-10) are efficient helper cells, TH1 cells also have
the capacity to be
helpers. (Paul, W. E., "Chapter 1: The immune system: an introduction,"
Fundamental
Immunology, 4th Edition, Ed. Paul, W. E., Lippicott-Raven Publishers,
Philadelphia (1999)).
T cells involved in Induction of Cellular Immunity
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[00119] T cells also may act to enhance the capacity of monocytes and
macrophages to destroy intracellular microorganisms. In particular, interferon-
gamma (IFN-
y) produced by helper T cells enhances several mechanisms through which
mononuclear
phagocytes destroy intracellular bacteria and parasitism including the
generation of nitric
oxide and induction of tumor necrosis factor (TNF) production. The TH1 cells
are effective
in enhancing the microbicidal action because they produce IFN-y. By contrast,
two of the
major cytokines produced by TH2 cells, IL-4 and IL-10, block these activities.
(Paul, W. E.,
"Chapter 1: The immune system: an introduction," Fundamental Immunology, 4th
Edition,
Ed. Paul, W. E., Lippicott-Raven Publishers, Philadelphia (1999)).
Suppressor or Regulatory T (Treg) cells
[00120] A controlled balance between initiation and downregulation of the
immune
response is important to maintain immune homeostasis. Both apoptosis and T
cell anergy (a
tolerance mechanism in which the T cells are intrinsically functionally
inactivated following
an antigen encounter (Scwartz, R. H., "T cell anergy," Annu. Rev. Immunol.,
21: 305-334
(2003)) are important mechanisms that contribute to the downregulation of the
immune
response. A third mechanism is provided by active suppression of activated T
cells by
suppressor or regulatory CD4+ T (Treg) cells. (Reviewed in Kronenberg, M. et
al.,
"Regulation of immunity by self-reactive T cells," Nature 435: 598-604
(2005)). CD4+
Tregs that constitutively express the IL-2 receptor alpha (IL-2RO ) chain
(CD4+ CD25+) are
a naturally occurring T cell subset that are anergic and suppressive. (Taams,
L. S. et 1.,
"Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and
apoptosis-
prone population," Eur. J. Immunol., 31: 1122-1131 (2001)). Depletion of
CD4+CD25+
Tregs results in systemic autoimmune disease in mice. Furthermore, transfer of
these Tregs
prevents development of autoimmune disease. Human CD4+CD25+ Tregs, similar to
their
murine counterpart, are generated in the thymus and are characterized by the
ability to
suppress proliferation of responder T cells through a cell-cell contact-
dependent mechanism,
the inability to produce IL-2, and the anergic phenotype in vitro. Human
CD4+CD25+ T
cells can be split into suppressive (CD25high) and nonsuppressive (CD25low)
cells,
according to the level of CD25 expression. A member of the forkhead family of
transcription
factors, FOXP3, has been shown to be expressed in murine and human CD4+CD25+
Tregs
and appears to be a master gene controlling CD4+CD25+ Treg development.
(Battaglia, M.
et al., "Rapamycin promotes expansion of functional CD4+CD25+Foxp3+ regulator
T cells
of both healthy subjects and type 1 diabetic patients," J. Immunol., 177: 8338-
8347 (200)).
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Cytotoxic T Lymphocytes (CTL)
[00121] The CD8+ T cells that recognize peptides from proteins produced within
the target cell have cytotoxic properties in that they lead to lysis of the
target cells. The
mechanism of CTL-induced lysis involves the production by the CTL of perforin,
a molecule
that can insert into the membrane of target cells and promote the lysis of
that cell. Perforin-
mediated lysis is enhanced by a series of enzymes produced by activated CTLs,
referred to as
granzymes. Many active CTLs also express large amounts of fas ligand on their
surface. The
interaction of fas ligand on the surface of CTL with fas on the surface of the
target cell
initiates apoptosis in the target cell, leading to the death of these cells.
CTL-mediated lysis
appears to be a major mechanism for the destruction of virally infected cells.
[00122] The term "matrix" as sued herein refers to a three dimensional network
of
fibers that contains voids (or "pores") where the woven fibers intersect. The
structural
parameters of the pores, including the pore size, porosity, pore
interconnectivity/tortuosity
and surface area, affect how fluid, solutes and cells move in and out of the
matrix.
[00123] The term "perfusion" as used herein refers to the process of nutritive
delivery of arterial blood to a capillary bed in biological tissue. Perfusion
("F") can be
calculated with the formula F=((PA-Pv)/R) wherein PA is mean arterial
pressure, Pv is mean
venous pressure, and R is vascular resistance. Tissue perfusion can be
measured in vivo, by,
for example, but not limited to, magnetic resonance imaging (MRI) techniques.
Such
techniques include using an injected contrast agent and arterial spin labeling
(ASL) (wherein
arterial blood is magnetically tagged before it enters into the tissue of
interest and the amount
of labeling is measured and compared to a control recording). Tissue perfusion
can be
measured in vitro, by, for example, but not limited to, tissue oxygen
saturation (St02) using
techniques including, but not limited to, hyperspectral imaging (HSI).
[00124] The terms "proliferation" and "propagation" are used interchangeably
herein to refer to expansion of a population of cells by the continuous
division of single cells
into identical daughter cells.
[00125] The term "polymer" as used herein refers to a macromolecule formedby
the chemical union of five or more identical combining units (monomers).
Exemplary
polymers by type include, without limitation, inorganic polymers (e.g.,
siloxane, sulfur
chains, black phosphorus, boron-nitrogen, aluminosilicate, borosilicate, or
boro-
aluminosilicate, glass ceramics, ceramics, and semiconductor or crystalline
materials (e.g.
silicones); Organic polymers, including natural organic polymers e.g.,
polysaccharides, such
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as starch, cellulose, pectin, seaweed gums (agar, etc), vegetable gums
(Arabic, etc.);
polypeptides (e.g., albumin, globulin); and hydrocarbons, e.g., polyisoprene;
synthetic
polymers, including thermoplastic polymers, such as polyvinyl chloride,
polyethylene
(linear), polystyrene, polypropylene, fluorocarbon resins, polyurethane, and
acrylate resins,
and thermosetting synthetic polymers, such as elastomers, polyethylene (cross-
linked),
penolics, and polyesters; and semisynthetic organic polymers, such as
cellulosics (e.g.,
methylcellulose, cellulose acetate) and modified starches. Further examples of
polymers
include, without limitation, hydrophilic polyethylene, polystyrenes,
polypropylenes,
acrylates, methacrylates, polycarbonates, polysulfones, polyesterketones, poly-
or cyclic
olefins, polychlorotrifluoroethylene, and polyethylene therephthalate.
[00126] The term "reduce" or "reducing" as used herein refers to the limiting
of an
occurrence of a disease, disorder or condition in individuals at risk of
developing the
disorder.
[00127] The term "relapse" as used herein refers to the return of a disease or
the
signs and symptoms of a disease after a period of improvement.
[00128] The term "Rho" as used herein refers to a subfamily of proteins
related to
the RAS subgroup thought to be involved in cell transformation and the
regulation of
morphology and function of dendritic cells. Non-limiting examples of Rho
proteins include
RhoA, RhoB and RhoC, RhoG ,RhoH, RhoQ, RhoU RhoV, Rnd 1, 2 and 3 (e.g., RhoE),
and
RAC1, 2, 3 and 4.
[00129] The term "ROCK"as used herein refers to Rho associated coil-coil
kinase.
ROCK proteins belong to the protein kinase A, G, and C family (AGC family) of
classical
serine/threonine protein kinases, a group that also includes other regulators
of cell shape and
motility, such as citron Rho-interacting kinase (CRIK), dystrophia myotonica
protein kinase
(DMPK), and the myotonic dystrophy kinase-related Cdc42-binding kinases
(MRCKs). The
main function of ROCK signaling is regulation of the cytoskeleton through the
phosphorylation of downstream substrates, leading to increased actin filament
stabilization
and generation of actin-myosin contractility. (Morgan-Fisher et al.,
"Regulation of ROCK
Activity in Cancer" (2013) 61:185-198, at 185).
[00130] Two homologous mammalian serine/threonine kinases, Rho-associated
protein kinases I and II (ROCK I and II), are key regulators of the actin
cytoskeleton acting
downstream of the small GTPase Rho. ROCK I (alternatively called ROK 0) and
ROCK II
(also known as Rho kinase or ROK a) are 160-kDa proteins encoded by distinct
genes. The
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GeneBank accession number for human ROCK I is EF445027.1; the GeneBank
accession
number for human ROCK II is NP 004841. The mRNA of both kinases is
ubiquitously
expressed, but ROCK I protein is mainly found in organs such as liver, kidney,
and lung,
whereas ROCK II protein is mainly expressed in muscle and brain tissue. The
two kinases
have the same overall domain structure and have 64% overall identity in
humans, with 89%
identity in the catalytic kinase domain. Both kinases contain a coiled-coil
region (55%
identity) containing a Rho-binding domain (RBD) and a pleckstrin homology (PH)
domain
split by a Cl conserved region (80% identity). Despite a high degree of
homology between
the two ROCKs, as well as the fact that they share several common substrates,
studies have
shown that the two ROCK isoforms also have distinct and non-redundant
functions. For
example, ROCK I has been shown to be essential for the formation of stress
fibers and focal
adhesions, whereas ROCK II is required for myosin II-dependent phagocytosis.
[00131] ROCKs exist in a closed, inactive conformation under quiescent
conditions, which is changed to an open, active conformation by the direct
binding of
guanosine triphosphate (GTP)-loaded Rho. (Morgan-Fisher et al., "Regulation of
ROCK
Activity in Cancer" (2013) 61:185-198). Rho is a small GTPase which functions
as a
molecular switch, cycling between guanosine diphosphate (GDP) and guanosine
triphosphate
(GTP) bound states under signaling through growth factors or cell adhesion
receptors.
(Morgan-Fisher et al., "Regulation of ROCK Activity in Cancer" (2013) 61:185-
198, at 185)
GTPases are hydrolase enzymes that bind and hydrolyze GTP. In a similar way to
ATP, GTP
can act as an energy carrier, but it also has an active role in signal
transduction, particularly in
the regulation of G protein activity. G proteins, including Rho GTPases, cycle
between an
inactive GDP-bound and an active GTP-bound conformation. The transition
between the two
conformational states occurs through two distinct mechanisms: activation by
GTP loading
and inactivation by GTP hydrolysis. GTP loading is a two-step process that
requires the
release of bound GDP and its replacement by a GTP molecule. Nucleotide release
is a
spontaneous but slow process that has to be catalyzed by RHO-specific guanine
nucleotide
exchange factors (RHOGEFs), which associate with RHO GTPases and trigger
release of the
nucleotide. The resulting nucleotide-free binary complex has no particular
nucleotide
specificity. However, the cellular concentration of GTP is markedly higher
than that of GDP,
which favors GTP loading, resulting in the activation of RHO GTPases.
[00132] Conversely, to turn off the switch, GTP has to be hydrolyzed. This is
facilitated by RHO-specific GTPase-activating proteins (RHOGAPs), which
stimulate the
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intrinsically slow hydrolytic activity of RHO proteins. Although guanine
nucleotide exchange
factors (GEFs) and GTPase-activating proteins (GAPs) are the canonical
regulators of this
cycle, several alternative mechanisms, such as post-translational
modifications, may fine-tune
the RHO switch. In addition, inactive RHO GTPases are extracted by RHO-
specific guanine
nucleotide dissociation inhibitors (RHOGDIs) from cell membranes to prevent
their
inappropriate activation and to protect them from misfolding and degradation.
(R. Garcia-
Mata et al. Nature Reviews Molecular Cell Biology (2011) 12:493-504; at 494).
[00133] Rho-ROCK signaling has been implicated in cell cycle regulation. Rho-
ROCK signaling increases cyclin D1 and Cipl protein levels, which stimulate
Gl/S cell cycle
progression. (Morgan-Fisher et al., "Regulation of ROCK Activity in Cancer"
(2013)
61:185-198, at 189). Polyploidization naturally occurs in megakaryocytes due
to an
incomplete mitosis, which is related to a partial defect in Rho-ROCK
activation, and leads to
an abnormal contractile ring lacking myosin IIA.
[00134] Rho-ROCK signaling also has been linked to apoptosis and cell
survival.
During apoptosis, ROCK I and ROCK II are altered to become constitutively-
active kinases.
Through proteolytic cleavage by caspases (ROCK I) or granzyme B (ROCK II), a
carboxyl-
terminal portion is removed that normally represses activity. Interaction with
phosphatidyl
inositol (3,4,5)-triphosphate (PIP3) provides an additional regulatory
mechanism by
localizing ROCK II to the plasma membrane where it can undertake spatially
restricted
activities, i.e. the regulation by localization of enzymatic activity.
Phosphorylation at
multiple specific sites by polo-like kinase 1 was found to promote ROCK II
activation by
RhoA. (Olson (2008) "Applications for ROCK kinase inhibition" Curr Opin Cell
Biol 20(2):
242-248, at 242.) Additional Serine/Threonine and Tyrosine kinases may also
regulate
ROCK activity given that more phosphorylations have been identified. (Olson
(2008)
"Applications for ROCK kinase inhibition" Curr. Opin. Cell Biol. 20(2): 242-
248, at 242.)
Specifically, protein oligomerization induces N-terminal trans-
phosphorylation. (K. Riento
and A.J. Ridley, "ROCKs: multifunction kinases in cell behavior." Nat. Rev.
Mol. Cell Biol.
(2003) 4:446-456) Other direct activators include intracellular second
messengers such as
arachodonic acid and sphingosylphosphorylcholine which can activate ROCK
independently
of Rho. Furthermore, ROCK1 activity can be induced during apoptosis. (Mueller,
B.K. et al.,
"Rho Kinase, a promising drug target for neurological disorders." (2005) Nat.
Rev. Mol. Cell
Biol. 4(6): 387-398.)
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[00135] ROCK protein signaling reportedly acts in either a pro- or anti-
apoptotic
fashion depending on cell type, cell context and microenvironment. For
instance, ROCK
proteins are essential for multiple aspects of both the intrinsic and
extrinsic apoptotic
processes, including regulation of cytoskeletal-mediated cell contraction and
membrane
blebbing, nuclear membrane disintegration, modulation of Bc12-family member
and caspase
expression/activation and phagocytosis of the fragmented apoptotic bodies
(Figure 4) (B.K.
Mueller et al. "Rho Kinase, a promising drug target for neurological
disorders." (2005)
Nature Rev.: Drug Discovery 4:387-398). In contrast, ROCK signaling also
exhibits pro-
survival roles (Figure 4). Though a wealth of data exists to suggest both pro-
and anti-
survival roles for ROCK proteins, the molecular mechanisms that modulate these
pleitropic
roles are largely unknown. (C.A. Street and B.A. Bryan, "Rho Kinase proteins ¨
pleiotropic
modulators of cell survival and apoptosis." (2011) Anticancer Res. 31(11):3645-
3657).
[00136] The term "risk of intestinal graft-versus-host disease (GVHD)" as used
herein refers to a potential for a population of T lymphocytes derived from a
potential
allogeneic donor to damage intestinal epithelial cells of a recipient subject.
[00137] The term "ROCK inhibitor" as used herein refers to any molecule that
decreases the function of a ROCK protein.
[00138] The term "stimulate" in any of its grammatical forms as used herein
refers
to inducing activation or increasing activity.
[00139] As used herein, the terms "subject" or "individual" or "patient" are
used
interchangeably to refer to a member of an animal species of mammalian origin,
including
humans. The term "a subject in need thereof' is used to refer to a subject in
need of allo-
BMT or a subject at risk for the complication GVHD disease.
[00140] The term "suspension culture" as used herein refers to cells which do
not
require attachment to a substratum to grow, i.e. they are anchorage
independent. Cell cultures
derived from blood are typically grown in suspension. Cells can grow as single
cells or
clumps. To subculture the cultures which grow as single cells they can be
diluted. However,
the cultures containing clumps need to have the clumps disassociated prior to
subculturing of
the culture.
[00141] The term "symptom" as used herein refers to a phenomenon that arises
from and accompanies a particular disease or disorder and serves as an
indication of it.
[00142] The term "syndrome," as used herein, refers to a pattern of symptoms
indicative of some disease or condition.
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[00143] The term "therapeutic agent" as used herein refers to a drug,
molecule,
nucleic acid, protein, metabolite, composition or other substance that
provides a therapeutic
effect. The term "active" as used herein refers to the ingredient, component
or constituent of
the compositions of the described invention responsible for the intended
therapeutic effect.
The terms "therapeutic agent" and "active agent" are used interchangeably
herein. The term
"therapeutic component" as used herein refers to a therapeutically effective
dosage (i.e., dose
and frequency of administration) that eliminates, reduces, or prevents the
progression of a
particular disease manifestation in a percentage of a population. An example
of a commonly
used therapeutic component is the ED50 which describes the dose in a
particular dosage that
is therapeutically effective for a particular disease manifestation in 50% of
a population.
[00144] The terms "therapeutic amount", "therapeutically effective amount", an
"amount effective", or "pharmaceutically effective amount" of an active agent
is used
interchangeably to refer to an amount that is sufficient to provide the
intended benefit of
treatment.
[00145] The term "therapeutic effect" as used herein refers to a consequence
of
treatment, the results of which are judged to be desirable and beneficial. A
therapeutic effect
can include, directly or indirectly, the arrest, reduction, or elimination of
a disease
manifestation. A therapeutic effect can also include, directly or indirectly,
the arrest
reduction or elimination of the progression of a disease manifestation.
[00146] General principles for determining therapeutic effectiveness, which
may
be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of
Therapeutics,
10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference,
are
summarized below.
[00147] Pharmacokinetic principles provide a basis for modifying a dosage
regimen to obtain a desired degree of therapeutic efficacy with a minimum of
unacceptable
adverse effects. In situations where the drug's plasma concentration can be
measured and
related to the therapeutic window, additional guidance for dosage modification
can be
obtained.
[00148] Drug products are considered to be pharmaceutical equivalents if they
contain the same active ingredients and are identical in strength or
concentration, dosage
form, and route of administration. Two pharmaceutically equivalent drug
products are
considered to be bioequivalent when the rates and extents of bioavailability
of the active
ingredient in the two products are not significantly different under suitable
test conditions.
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[00149] The term "therapeutic window" refers to a concentration range that
provides therapeutic efficacy without unacceptable toxicity. Following
administration of a
dose of a drug, its effects usually show a characteristic temporal pattern. A
lag period is
present before the drug concentration exceeds the minimum effective
concentration ("MEC")
for the desired effect. Following onset of the response, the intensity of the
effect increases as
the drug continues to be absorbed and distributed. This reaches a peak, after
which drug
elimination results in a decline in the effect's intensity that disappears
when the drug
concentration falls back below the MEC. Accordingly, the duration of a drug's
action is
determined by the time period over which concentrations exceed the MEC. The
therapeutic
goal is to obtain and maintain concentrations within the therapeutic window
for the desired
response with a minimum of toxicity. Drug response below the MEC for the
desired effect
will be subtherapeutic, whereas for an adverse effect, the probability of
toxicity will increase
above the MEC. Increasing or decreasing drug dosage shifts the response curve
up or down
the intensity scale and is used to modulate the drug's effect. Increasing the
dose also prolongs
a drug's duration of action but at the risk of increasing the likelihood of
adverse effects.
Accordingly, unless the drug is nontoxic, increasing the dose is not a useful
strategy for
extending a drug's duration of action.
[00150] Instead, another dose of drug should be given to maintain
concentrations
within the therapeutic window. In general, the lower limit of the therapeutic
range of a drug
appears to be approximately equal to the drug concentration that produces
about half of the
greatest possible therapeutic effect, and the upper limit of the therapeutic
range is such that
no more than about 5% to about 10% of patients will experience a toxic effect.
These figures
can be highly variable, and some patients may benefit greatly from drug
concentrations that
exceed the therapeutic range, while others may suffer significant toxicity at
much lower
values. The therapeutic goal is to maintain steady-state drug levels within
the therapeutic
window. For most drugs, the actual concentrations associated with this desired
range are not
and need not be known, and it is sufficient to understand that efficacy and
toxicity are
generally concentration-dependent, and how drug dosage and frequency of
administration
affect the drug level. For a small number of drugs where there is a small (two-
to three-fold)
difference between concentrations resulting in efficacy and toxicity, a plasma-
concentration
range associated with effective therapy has been defined.
[00151] In this case, a target level strategy is reasonable, wherein
a desired
target steady-state concentration of the drug (usually in plasma) associated
with efficacy and
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minimal toxicity is chosen, and a dosage is computed that is expected to
achieve this value.
Drug concentrations subsequently are measured and dosage is adjusted if
necessary to
approximate the target more closely.
[00152] In
most clinical situations, drugs are administered in a series of repetitive
doses or as a continuous infusion to maintain a steady-state concentration of
drug associated
with the therapeutic window. To maintain the chosen steady-state or target
concentration
("maintenance dose"), the rate of drug administration is adjusted such that
the rate of input
equals the rate of loss. If the clinician chooses the desired concentration of
drug in plasma
and knows the clearance and bioavailability for that drug in a particular
patient, the
appropriate dose and dosing interval can be calculated.
[00153] The term "two-dimensional tissue construct" as used herein refers to a
collection of cells and the intercellular substances surrounding them in a
geometric
configuration having length and width.
[00154] The term "three-dimensional tissue construct" as used herein refers to
a
tissue like collection of cells and the intercellular substances surrounding
them in a geometric
configuration having length, width, and depth.
[00155] The term "transplantation" as used herein, refers to removal and
transfer
of cells, a tissue or an organ from one part or individual to another.
[00156] As used herein the term "treating" includes abrogating, substantially
inhibiting, slowing or reversing the progression of a condition, substantially
ameliorating
clinical symptoms of a condition, or substantially preventing the appearance
of clinical
symptoms of a condition. Treating further refers to accomplishing one or more
of the
following: (a) reducing the severity of the disorder; (b) limiting development
of symptoms
characteristic of the disorder(s) being treated; (c) limiting worsening of
symptoms
characteristic of the disorder(s) being treated; (d) limiting recurrence of
the disorder(s) in
patients that have previously had the disorder(s); and (e) limiting recurrence
of symptoms in
patients that were previously asymptomatic for the disorder(s).
[00157] The term "tumor necrosis factor" (TNF) as used herein refers to a
cytokine
made by white blood cells in response to an antigen or infection, which induce
necrosis
(death) of tumor cells and possesses a wide range of pro-inflammatory actions.
Tumor
necrosis factor also is a multifunctional cytokine with effects on lipid
metabolism,
coagulation, insulin resistance, and the function of endothelial cells lining
blood vessels.
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[00158] The terms "VEGF-1" or "vascular endothelial growth factor-1" are used
interchangeably herein to refer to a cytokine that mediates numerous functions
of endothelial
cells including proliferation, migration, invasion, survival, and
permeability. VEGF is critical
for angiogenesis.
I. An Ex Vivo Model Of Persistent T Lymphocyte Stimulation Events
Encountered
By Circulating T Lymphocytes At Lymph Node And Tissue Levels In Vivo
Comprising
A Multiwell Plate-Based Perfusion Culture Device
[00159] According to one aspect, an ex vivo model of persistent T lymphocyte
stimulation events encountered by circulating T lymphocytes at lymph node and
tissue levels
in vivo comprises
[00160] (a) A multiwell plate-based perfusion culture device, comprising, from
top
to bottom:
[00161] a bottomless multi-well plate comprising a plurality of bottomless
wells;
[00162] a first micropatterned polymer layer attached to a bottom surface of
the
bottomless multi-well plate to form a plurality of adjacent wells, one or more
of each pair of
adjacent wells comprising a transparent polymer membrane placed within the one
ore mover
of each pair of adjacent wells;
[00163] a second micropatterned polymer layer comprising two or more holes
that
correspond to two or more adjacent wells, the second micropatterned polymer
layer being
attached to a bottom surface of the first micropatterned polymer layer, such
that each hole of
the second micropatterned polymer layer is aligned with the two or more
adjacent wells in
the first micropatterned polymer layer, one or more of each pair of adjacent
wells comprising
the transparent polymer membrane;
[00164] a microfluidic channel formed between the two adjacent wells that
allows
internal fluidic communication between the two adjacent wells;
[00165] one or more removable polymer plugs, each located at a top surface of
each of the plurality of wells, and one or more tubes, each connected to the
one or more
polymer plugs;
[00166] a pump connected to a reservoir that removably connects to the tubes;
[00167] a transparent, optical grade glass layer attached to the bottom
surface of
the second micropatterned polymer layer that forms a bottom surface for the
plurality of wells
and that seals the multi-well plate perfusion culture device;
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[00168] wherein one or more of the two adjacent wells is a culture chamber for
culturing a population of cells;
[00169] (b) an expanded population of cells derived from a recipient subject
comprising a cell-specific antigen in the first adjacent well of the device in
(a);
[00170] (c) an expanded population of T lymphocytes derived from a potential
donor of a BMT graft in the second adjacent well of the device in (a), wherein
the potential
donor is allogeneic to the recipient subject;
[00171] (d) A liquid culture medium that is flowable between the first
adjacent
well and the second adjacent well;
[00172] The model being characterized by:
[00173] (i) Circulation of the liquid medium from the first well into the
second
well and back to the first well through the microfluidic channel;
[00174] (ii) An interaction between the population of cells comprising the
cell
antigen derived from the recipient subject in the first well and the
population of T
lymphocytes is effective to generate alloreactive effector T lymphocytes; and
[00175] (iii)Alloreactive effector T lymphocyte-induced quantifiable damage to
the
population of cells comprising the cell antigen derived from the recipient by
the population of
alloreactive effector T lymphocytes from the donor allogeneic to the
recipient.a multiwell
plate-based perfusion culture device for culturing cells ex vivo with
continuous perfusion of a
liquid medium is used to replicate the interactions of human T cells and
intestinal epithelial
cells in vivo.
[00176] With respect to the multiwell plate-based perfusion culture device,
according to one embodiment of the described invention, the device comprises a
plurality of
layers. According to some such embodiments, the multiwall plate-based
perfusion culture
device comprises a bottomless multi-well plate including a plurality of
bottomless wells; a
first micropatterned polymer layer comprising a plurality of transparent
polymer membranes
therein, a second micropatterned polymer layer comprising a plurality of holes
therethrough,
a third micropatterned polymer layer comprising a plurality of holes
therethrough, one blank
glass layer for use with plate readers; and a plurality of fluidic passages
formed between the
polymer membrane and the blank glass layer. The term "bottomless multi-well
plate" as used
herein refers to a multi-well plate without a bottom surface; and the term
"bottomless wells"
as used herein refers to wells of the multi-well plate without a bottom
surface.
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[00177] According to some embodiments, the device can comprise more than three
micropatterned polymer layers.
[00178] According to some embodiments, the first micropatterned polymer layer
is
attached to a bottom surface of the bottomless multi-well plate such that each
of the plurality
of transparent polymer membranes corresponds to each of the plurality of wells
when the
number of the polymer membranes is equal to the number of the wells, wherein
the second
micropatterned polymer layer is attached to a bottom surface of the first
micropatterned
polymer layer such that each of the plurality of holes corresponds to each of
the plurality of
wells.
[00179] According to some embodiments, a polymer membrane is placed in every
other well in the multiwell plate so that the number of the polymer membranes
in the
multiwall plate equals one-half of the number of the wells.
[00180] According to some embodiments, the third micropatterned polymer layer
is attached to a bottom surface of the second micropatterned polymer layer
such that each of
the plurality of holes in the third micropatterned polymer layer corresponds
to two adjacent
wells, thereby creating a microfluidic channel between the two adjacent wells
to allow
internal fluidic communication between the two adjacent wells. According to
some
embodiments, the third micropatterned polymer layer is attached to a bottom
surface of the
second micropatterned polymer layer such that each of the plurality of holes
in the third
micropatterned polymer layer corresponds to more than two adjacent holes in
the second
micropatterned polymer layer.
[00181] According to some embodiments, the second micropatterned polymer
layer is omitted, and the third micropatterned polymer layer is attached to
the bottom surface
of the first micropatterned polymer layer, such that each hole of the third
micropatterned
polymer layer corresponds to one or more adjacent polymer membranes in the
first
micropatterned polymer layer.
[00182] According to some embodiments, the microfluidic channel is 200 p.m
thick
and 5 p.m high.
[00183] According to some embodiments, one of the two adjacent wells is a
culture
chamber, which is used to culture cells or tissues; and the second adjacent
well is an outlet
chamber, which is used to direct the effluent streams to exit through the top
of the device,
wherein a first tubing attached to the culture chamber is an inlet and a
second tubing attached
to the outlet chamber is merely an outlet, thus providing re-circulation of
liquid medium
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together with non-adherent cells between two chambers. According to some such
embodiments, the outlet chamber may or may not contain a polymer membrane.
[00184] According to some embodiments, both of the two adjacent wells are
culture chambers, which are used to culture different cells or tissues. For
example, according
to an embodiment wherein both of the two adjacent wells are culture chambers,
and these
culture chambers are used to screen samples for determining a patient's risk
of developing
GVHD, the first chamber is used to culture epithelial cells, and the second is
used to culture
dendritic cells. According to some embodiments, the tubing connected to the
first culture
chamber is an inlet and another tubing connected to the second culture chamber
is an outlet;
thus providing re-circulation of liquid medium together with non-adherent
cells between the
two chambers.
[00185] According to some embodiments, the blank glass layer provides optical
access through the bottom of the chambers for cell characterization with plate
readers.
According to some embodiments, the blank glass layer is attached to a bottom
of the third
micropatterned polymer layer to seal the multi-well plate culture device
thereby forming a
bottom surface thereof for the plurality of wells. According to some
embodiments, the blank
glass layer is about 1.2 mm-thick.
[00186] According to some embodiments, instead of comprising a plurality of
layers, the well plate-based perfusion culture device comprises one polymer
substrate which
has multiple layers of holes therein, a first layer of holes comprises a
plurality of holes, each
corresponding to a shape and size and location of each of the plurality of
wells, and a second
layer of holes comprises a plurality of holes, each corresponding to a size
and location of
every two adjacent wells, thereby allowing internally fluidly connection
between every two
adjacent wells. According to some embodiments, each of the plurality of holes
in the first
layer of polymer substrate further has a transparent polymer membrane attached
thereto.
[00187] According to some embodiments, the polymer substrate is made from
polymer extrusion molding.
[00188] According to some embodiments, the micropatterned polymer layers are
made of a polymer, e.g., polydimethyl siloxane (PMDS), polystyrene or the
like.
[00189] According to some embodiments, the multi-well plate, the
micropatterned
polymer layers, and the glass layer are bonded (meaning joined securely to
each other, for
example, by an adhesive, a heat process, or pressure) using oxygen plasma
treatments.
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[00190] According to some embodiments, the multi-well plate-based perfusion
culture device further comprises a plurality of removable polymer plugs
(meaning a piece of
material used to stopper an aperture), each located at a top surface of each
of the plurality of
wells; and a plurality of tubes (meaning a hollow, elongated body), each
connected to each of
the plurality of polymer plugs. According to some embodiments, the removable
polymer
plugs are made of a polymer, e.g., PDMS, polystyrene, or the like. According
to some such
embodiments, the removable polymer plugs made of PDMS are made by soft
lithography.
According to some such embodiments, the removable polymer plugs made of
polystyrene
(PS) are made by PS extrusion and bonding.
[00191] According to some embodiments, the device further comprises at least
one
pump connected to at least one reservoir, which removably connects to the
tubes, e.g., the
first tube and the second tube. According to some such embodiments; the pump
controls
flow rate of recirculation of the liquid medium, for example, via one or more
valves, into and
out of the wells.
[00192] According to some embodiments, the tube that connects the two adjacent
chambers at the top of the device is a U-shaped tubing, and flow of a liquid
medium is driven
by the difference between an amount of liquid medium inside chamber 1 and
chamber 2 until
equilibrium is established.
[00193] According to some embodiments, a method for culturing cells in the
multiwall plate device comprises (a) providing a liquid medium into a first
well that is fluidly
connected to a second well, such that the liquid medium flows from the first
well into the
second well, which is the well adjacent to the first well through the
microfluidic channel, and
(2) recycling the liquid medium back to the first well through a reservoir and
pump or a U-
tube externally connecting the two wells at the top of the device. According
to some
embodiments, the liquid medium flows at a rate of about 10-50 .tt/min.
According to some
embodiments, the multi-well plate comprises at least 6, at least 12, at least
24, at least 48, at
least 96, at least 384 or at least 1536 wells. The wells may have dimensions
substantially
same as the dimensions of the wells in plate currently commercially available
for
commercially available readers and dispensers. According to some embodiments,
the multi-
well plate has a substantially rectangular shape appropriate for commercially
available
readers and dispensers. According to some embodiments, the multi-well plate
can have a
shape different from rectangular.
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[00194] According to some embodiments, the multi-well plate may be constructed
of polymeric materials. Exemplary polymers include, without limitation,
hydrophilic
polyethylenes, polystyrenes, polypropylenes, acrylates, methacrylates,
polycarbonates,
polysulfones, polyesterketones, poly- or cyclic olefins,
polychlorotrifluoroethylene, and
polyethylene therephthalate. According to some embodiments, the multi-well
plate can be
constructed of polystyrene. According to some embodiments, the multi-well
plate may be
constructed of inorganic polymer materials.
[00195] According to some embodiments, the transparent polymer membrane
provides optical access through the bottom surface of the culture chambers for
cell
characterization with plate readers. According to some embodiments, the
transparent
polymer membrane anchors tissue cells and biomaterials. According to some
embodiments,
the transparent polymer membrane is a transparent polycarbonate (PC) membrane.
According to some embodiments, the transparent polymer membrane is a
polyethylene
terephthalate (PET) membrane. According to some such embodiments, the PET
membrane
has an average pore size of 8 p.m.
[00196] According to some embodiments, the micropatterned polymer layers are
used to anchor placement of the polymer membranes within the wells of the
device that
comprise one or more culture chambers.
[00197] According to some embodiments, the micropatterned polymer layers are
constructed of a polymer. According to some such embodiments, the
micropatterned
polymer layers are made of polydimethyl siloxane (PMDS) or polystyrene.
According to
some such embodiments, the micropatterned polymer layers made of PMDS are made
by soft
lithography. According to some such embodiments, the micropatterned polymer
layers made
of polystyrene (PS) are made by PS extrusion and bonding.
[00198] According to some embodiments, the device further comprises
biocompatible non-living material formed into a three-dimensional structure
comprising
interstitial spaces, for example, nanofibers or microbeads that are placed on
a top surface of
the polymer membrane. According to some embodiments, the microbeads comprise a
polymer. According to some such embodiments, the microbeads comprise
polystyrene.
According to some such embodiments, the microbeads comprise biphasic calcium
phosphate
(BCP).
[00199] According to some embodiments, the polymer membrane is coated with a
nanofiber mesh. According to some such embodiments, the nanofiber mesh
comprises an
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electrospun PCL/collagen mesh. According to some embodiments, the PCL/collagen
mesh
comprises a nanofiber matrix comprising a plurality of pores through which the
population of
T lymphocytes derived from the potential donor allogeneic to the recipient
subject can pass.
According to some embodiments, the nanofiber matrix comprising the plurality
of pores
mimics the basement membrane of epithelial tissue and supports viability of
the intestinal
epithelial cells derived from the recipient subject.
[00200] According to some embodiments, the population of T lymphocytes in (c)
is
derived from peripheral blood lymphocytes of the potential donor. According to
some
embodiments, the population of T lymphocytes comprises a suspension of
nonadherent cells.
According to some embodiments, wherein the suspension of nonadherent cells
contains T
lymphocytes derived from the allogeneic donor. According to some embodiments,
wherein
the population of alloreactive effector T lymphocytes comprises alloreactive
activated antigen
presenting cells. According to some embodiments, wherein the alloreactive
activated antigen
presenting cells comprise a population of alloreactive activated dendritic
cells. According to
some embodiments, wherein the quantifiable damage to the population of cells
comprising
the cell-specific antigen derived from the recipient induced by the population
of nonadherent
alloreactive effector T lymphocytes from the donor allogeneic to the recipient
comprises cell
death. According to some embodiments, wherein the population of cells
comprising the
cell-specific antigen in (b) are a population of intestinal epithelial cells
derived from the
recipient subject, and the alloreactive effector T lymphocyte-induced
quantifiable damage to
the population of intestinal epithelial cells of the recipient subject is an
ex vivo measure of
risk of graft vs. host disease.
II. A Method For Optimizing Donor Selection For Allogeneic Transplantation
And
For Predicting Risk Of GVHD
[00201] A method for optimizing donor selection for allogeneic blood and
marrow
transplantation (BMT) therapy comprises, in order:
[00202] (a) acquiring a tissue sample from a recipient subject allogeneic to a
potential donor of a BMT graft, the tissue sample comprising a population of
primary
intestinal epithelial cells comprising an intestinal epithelial cell-specific
antigen;
[00203] (b)
seeding the population of primary intestinal epithelial cells of (a) in a
first adjacent well of a multiwall plate-based perfusion culture device, the
first adjacent well
comprising a transparent polymer membrane, expanding the population in a first
liquid
medium containing ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2
fibroblast
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feeder layer and generating a population of conditional reprogrammed
intestinal epithelial
cells (CRIECs) comprising the intestinal cell-specific antigen derived from
the recipient
subject;
[00204] (c) acquiring a population of T lymphocytes from the potential donor
allogeneic to the recipient;
[00205] (d) seeding and expanding in a second adjacent well of the multiwall
plate-based perfusion culture device the population of T lymphocytes derived
from the
potential donor of (c),
[00206] (e) co-culturing in a second liquid medium the CRIECs derived from the
recipient subject in the first adjacent well and the T lymphocytes derived
from the potential
donor allogeneic to the recipient subject in the second adjacent well, the co-
culturing being
characterized by:
[00207] (i) the first adjacent well being fluidly connected to the second
adjacent
well so that the second liquid medium is flowable between the first adjacent
well and the
second adjacent well; and
[00208] (ii) an interaction between the population of CRIECs derived from the
recipient subject and the population of T lymphocytes that is effective to
generate alloreactive
effector T lymphocytes derived from the potential allogeneic donor;
[00209] (f) measuring damage to the population of CRIECs derived from the
recipient subject induced by the alloreactive effector T lymphocytes derived
from the
potential donor allogeneic to the recipient subject, wherein the damage is a
measure of a risk
of intestinal graft versus host disease in the recipient subject;
[00210] (g) ranking a plurality of potential donors by the measure of the risk
of
intestinal graft versus host disease; and
[00211] (h) treating the recipient subject with a BMT graft derived from a
selected
donor allogeneic to the recipient subject whose T lymphocytes are
characterized by a reduced
risk of intestinal graft-versus-host disease.
[00212] According to some embodiments, the sample is a biopsy sample.
According to some embodiments, the biopsy sample is a small biopsy sample of
the order of
3 mm3. According to some embodiments, the biopsy sample is collected from
intestinal
tissue. According to some embodiments, the biopsy sample is collected from
intestinal tissue
by colonoscopy, endoscopy, or a combination thereof.
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[00213] According to some embodiments the potential donor is a haploidentical
donor (i.e., parent, child and other close relative),
[00214] According to some embodiments, the patient sample is acquired soon
after
diagnosis of a hematological malignancy for which allogeneic BMT is a
potential therapeutic
approach and stored for later use in the method. According to some
embodiments, the patient
sample is acquired in the relapse setting after chemotherapeutic interventions
have been
exhausted.
[00215] According to some embodiments, any cells of interest may be cultured.
According to some embodiments, the cells to be cultured can include normal,
diseased, stem,
cancerous, and/or mutated cells.
[00216] According to some embodiments, the primary IECs are prepared from the
small intestine, large intestine or colon of the recipient subject, and
expanded using
conditional reprogrammed cell technology, which comprises cultivating the
primary IECs in
a medium containing ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2
fibroblast
feeder layer. According to some embodiments the medium for cultivating the
primary
human IECs containing ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2
fibroblast
feeder layer is RPMI.
[00217] While expansion of conditionally reprogrammed cells is useful in
expanding the IECs from biopsy samples, CRCs cannot be used for co-culture of
CRIECs
and T cells for 2-3 weeks due to adverse effects of CRC media additives (e.g.,
ROCK kinase
inhibitor) on T cell motility and functionality. (Riento, et al., Molecular
cell biology (2003) 4,
446-456; Iyengar, et al., Journal of the American Society for Blood and Marrow
Transplantation, doi:10.1016/j.bbmt.2014.04.029 (2014)). According to some
embodiments,
the CRC medium is replaced with a complete RPI-1640 medium (defined as RPMI-
1640
supplemented with 10% fetal bovine serum and 5% L-glutamine) to culture the T
cells.
[00218] In native tissues, IECs reside on the thin fibrous basement membrane
(BMa) consisting of intermingled networks of laminins and type IV collagen and
provide cell
anchoring and barrier functions. The membrane networks interact with cells
through
membranous integrin receptors and other plasma membrane molecules, influencing
cell
differentiation, migration, adhesion, phenotype and survival.
[00219] According to some embodiments, the first well comprises a nanofibrous
coated transparent polymer membrane. According to some embodiments, the
nanofibrous
coating is prepared by electrospinning. According to some embodiments, the
nanofibrous
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coating comprises a fiber matrix of polycaprolactone in which extracellular
matrix (ECM)-
like molecules (e.g., collagen) is dispersed. According to some embodiments,
the
nanofibrous coated transparent polymer membrane is effective to maintain the
long term
functionality of CRIECs and T cells using RPMI as a common culture medium.
Wang's
prior research (Fu, et al. Biomaterials (2014) 35, 1496-1506) has shown that,
as a result of
mimicking the morphological and dimensional characteristics of base membrane
extracellular
matrix (ECM) fibrils, nanofibrous meshes can support keratinocytes to form
skin-like
structures and maintain cobble stone-like morphology for 2 weeks (Huang, et
al.,
Biomaterials (2012) 33, 1791-1800).
[00220] According to some embodiments, the method comprises providing
polymer microbeads preconditioned with one or more adhesion-promoting agents
to promote
adhesion of cells to at least one surface of the microbeads. According to some
such
embodiments, the cells are dendritic cells (DCs). According to some
embodiments, the
adhesion promoting agent comprises an effective amount of lipopolysaccharides
(LPS),
wherein the LPS are effective to promote adhesion of the DCs to the microbeads
surface.
[00221] According to some embodiments, the first well of the multiwall plate
device contains a population of conditionally reprogrammed IECs prepared from
a
mammalian subject, and the second well fluidly connected to the first well
contains T cells
comprising dendritic cells from a mammal allogeneic to the mammalian subject.
According
to some such embodiments, the mammal is a mouse. According to some such
embodiments,
the mammal is a human.
[00222] According to some embodiments, a CRIEC culture chamber can be
established by placing CRIECs into the first well on top of a polymer membrane
coated with
an electrospun PCL/collagen nanofibrous mesh.
[00223] According to some embodiment, an average open space (or pore size) in
the nanofibrous mesh is within a range of about 1-10 p.m. According to some
such
embodiments, the average open space (or pore size) in the nanofiber mesh is
about 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 p.m.
[00224] According to some embodiments, an antigen presenting cell (APC)
culture
chamber can be established by placing T cells comprising DCs in the liquid
medium onto the
pre-treated polymer microbeads in the second well. According to some
embodiments, one or
more cytokines can be added to the culture chamber to prolong T cell
maintenance.
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According to one embodiment, the population of T-cells suspended in the liquid
medium
comprises about 105 to 106 cells.
[00225] According to some embodiments, the method further comprises
replenishing DCs with new DCs by opening a polymer plug on the top of the APC
chamber
and placing new DCs onto the top of the microbead assembly. According to some
embodiments, the dendritic cell assembly can be replaced by a new
microbead/dendritic cell
assembly.
[00226] According to some embodiments, an average size of a polymer microbead
is in a range of about 45-90 p.m. According to some such embodiments, the
average size of a
polymer microbead is about 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 p.m.
[00227] According to some embodiments, the T cells comprising dendritic cells
are
derived from peripheral blood lymphocytes. According to some embodiments,
mouse
dendritic cells are enriched by injecting host mice with a B16-FLt3L tumor.
[00228] According to some embodiments, the polymer membrane has an
average
pore size that provides a sufficient opening for T cells to go through.
According to some
such embodiments, the average pore size of the polymer membrane is about 7-13
p.m.
According to some such embodiments, the average pore size of the polymer
membrane is
about 7, 8, 9, 10, 11, 12 or 13 p.m. According to some such embodiments, an
average
diameter of a T cell is about 5 p.m.
[00229] According to some embodiments, the nanofibrous coated transparent
polymer membrane is effective to anchor a population of cells. According to
some
embodiments, the polymer membrane comprises the population of human intestinal
epithelial
cells, the population of CRIECs, or a combination thereof.
[00230] According to some embodiments the cells to be cultured can be cultured
in
free suspensions, encapsulated in suitable hydrogels, encapsulated in
matrices, and/or
encapsulated in scaffolds. For example, according to some embodiments, the T
cells
comprising a suspension of about 106 T cells (e.g., eGFP m-T cells (harvested
from an eGFP
transgenic B6 mouse) or h-T cells) in a culture medium are flowable, i.e.,
they circulate with
the liquid medium of the microfluidic well plate-based perfusion culture
device. According
to one embodiment, the culture medium contains retinoic acid, which
facilitates the
generation of T cells with superior IEC-killing avidity.
[00231] According to some embodiments, a ratio of CRIECs: T cells is in a
range
of from 2:1 to 20:1. According to some such embodiments, the ratio of CRIECS:
T cells is
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2:1, 34:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,
16:1, 17:1, 18:1, 19:1,
or 20:1.
[00232] According to some embodiments, a ratio of T cells: DCs is in a range
of
from 1:1 to 20:1. According to some such embodiments, the ratio of T cells:
DCs is 1:2, 2:1,
3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12: 1, 13:1, 14:1, 15:1, 16:1,
17:1, 18: 1, 19:1, or
20:1. According to some such embodiments, the ratio of T cells: DCs is 5:1.
[00233] According to some embodiments, the co-culturing of the population of
human IECs from the proposed recipient subject and the population of T
lymphocytes from
the donor allogeneic to the recipient subject is effective to generate
alloreactive activated T-
lymphocytes. According to some embodiments, the allogeneic activated/effector
T cells
recirculate through interconnected h-CRIEC and antigen presenting cell (APC)
culture
chambers. According to some embodiments, the alloreactive activated/effector T-
lymphocytes comprise a population of antigen presenting cells. According to
some such
embodiments, the population of antigen presenting cells comprises a population
of dendritic
cells. According to some embodiments, the alloreactive T cells become
activated by
cognitive alloantigens on h-CRIECs. According to some embodiments, the
alloreactive
activated/effectorT cells comprise activated antigen presenting cells (APCs).
According to
some embodiments, the APCs comprise activated/effector dendritic cells.
According to some
embodiments, the alloreactive activated/effector T cells are effective to
induce quantifiable
damage to the population of h-CRIECs. According to some such embodiments, the
device is
clinically viable, i.e., it is effective to increase the critical number of
functional T cells
required to induce quantifiable alloreactivity in the CRIEC culture chamber
within a
diagnostic screening time frame of 2-3 weeks.
[00234] According to some embodiments, quantifiable damage to the population
of
CRIECs comprises measurable killing of the population of CRIECS. According to
some
embodiments, a pathological index (PIdx) is used to quantify T cell induced
CRIEC damage.
For example, the predictive capability of co-culture killing assays can be
compared to known
in vivo outcomes from well-established murine models of BMT.
[00235] According to some such embodiments, a panel of cell death analysis
methods is used to quantify cell death. For example, annexin V/PI staining
using flow
cytometry and in situ detection of cleaved caspase-3 using immunofluorescence
can be used
to determine cell death. According to some embodiments, the percentage of dead
cells is
calculated as [% of Annexin V+/PI+ cells in co-cultures - % of Annexin V+/PI+
cells in IEC
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alone cultures] (flow cytometric measurement). According to some embodiments,
CD3
staining is performed to identify adherent T cells contributing to the
response. According to
some embodiments, the caspases-3 staining is conducted in the well plate to
determine cell
death as [fluorescence intensity in co-culture - fluorescence intensity in IEC
alone
cultures]/[fluorescence intensity of DAPI staining, as an indicator of the
number of nucleated
cells in the cultures].
[00236] According to some embodiments, cell death is evaluated at three or
more T
cell-IEC ratios (i.e., the effector: target, or E:T ratio). According to some
embodiments, the
E:T ratio is 30, 10, or 3. See Choksi, S. et al, "A cD8 DE loop peptide analog
prevents graft
versus host disease in a multiple minor histocompatibility antigen-mismatched
bone marrow
transplantation model," Biology of Blood and Marrow Transplantation: 10: 669-
680, doi:
10.1016/j.bbmt.2004.06.005 (2004)).
[00237] According to some embodiments, the PIdx can be determined as the slope
of the curve of percentage of dead cells vs. E:T ratios, where a steeper curve
indicates a
higher risk for developing GVHD. According to some embodiments, the PIdx can
be
determined at multiple time points post-co-culture.
[00238] According to some embodiments, the recipient subject and potential
donor
are mammals.
[00239] According to some embodiments, the recipient subject and potential
allogeneic donor are mice.
[00240] According to some embodiments, the recipient subject and potential
allogeneic donor are human.
[00241] According to some embodiments, the method further comprises
identifying donor T cell clones responsible for the damage to the population
of CRIECS, and
selectively deleting the specific donor T cells clones responsible for the
damage from the
population of donor T lymphocytes.
[00242] According to some embodiments, the method comprises dissociating the
donor T cell clones responsible for the damage to the population of CRIECS
from donor T
cell clones responsible for an anti-tumor specific T cell response, such that
a therapeutically
effective amount of the remaining donor T cell clones is effective to mediate
GVT responses
against tumor specific antigens.
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[00243] According to some embodiments, the cells are cultured only in the
first
well, and the connected adjacent second well is an outlet well providing exit
of the liquid
medium from the top of the device.
[00244] According to some embodiments, cells of different types may be
cultured
at the same time in different fluidly connected wells of the plate-based
perfusion device. For
example, a first cell type can be seeded in and cultured in the first well
while a second cell
type can be seeded in and cultured in the second well at the same time.
III. A method for minimizing risks and maximizing benefits of
performing
blood and marrow transplantation (BMT) in a recipient subject, wherein the
recipient
subject has a hematologic malignancy.
[00245] A method for minimizing risks and maximizing benefits of
performing
blood and marrow transplantation (BMT) in a recipient subject, wherein the
recipient subject
has a hematologic malignancy, comprising, in order:
(A) Evaluating a population of T lymphocytes derived from a
potential donor,
allogeneic to the recipient subject, for a potential to damage intestinal
epithelial cells of the recipient subject according to steps (a) through (g)
of the method above, i.e.,
[00246] (a) acquiring a tissue sample from a recipient subject allogeneic to a
potential donor of a BMT graft, the tissue sample comprising a population of
primary
intestinal epithelial cells comprising an intestinal epithelial cell-specific
antigen;
[00247] (b) seeding the population of primary intestinal epithelial
cells of (a) in a
first adjacent well of a multiwall plate-based perfusion culture device, the
first adjacent well
comprising a transparent polymer membrane, expanding the population in a first
liquid
medium containing ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2
fibroblast
feeder layer and generating a population of conditional reprogrammed
intestinal epithelial
cells (CRIECs) comprising the intestinal cell-specific antigen derived from
the recipient
subject;
[00248] (c) acquiring a population of T lymphocytes from the potential donor
allogeneic to the recipient;
[00249] (d) seeding and expanding in a second adjacent well of the multiwall
plate-based perfusion culture device the population of T lymphocytes derived
from the
potential donor of (c),
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[00250] (e) co-culturing in a second liquid medium the CRIECs derived from the
recipient subject in the first adjacent well and the T lymphocytes derived
from the potential
donor allogeneic to the recipient subject in the second adjacent well, the co-
culturing being
characterized by:
[00251] (i) the first adjacent well being fluidly connected to the second
adjacent
well so that the second liquid medium is flowable between the first adjacent
well and the
second adjacent well; and
[00252] (ii) an interaction between the population of CRIECs derived from the
recipient subject and the population of T lymphocytes that is effective to
generate alloreactive
effector T lymphocytes derived from the potential allogeneic donor;
[00253] (f) measuring damage to the population of CRIECs derived from the
recipient subject induced by the alloreactive effector T lymphocytes derived
from the
potential donor allogeneic to the recipient subject, wherein the damage is a
measure of a risk
of intestinal graft versus host disease in the recipient subject;
[00254] (g) ranking a plurality of potential donors by the measure of the risk
of
intestinal graft versus host disease; and;
[00255] (B) evaluating the population of T lymphocytes derived from the
potential
donor allogeneic to the recipient subject for an effective graft versus tumor
response against
the tumor-specific antigens by:
[00256] (i) acquiring a specimen comprising a population of tumor cells
derived
from the recipient subject, the population of tumor cells comprising one or
more tumor
specific antigens;
[00257] (ii) seeding and expanding the population of tumor cells in the second
liquid medium in a third adjacent well of the multiwell plate-based perfusion
culture device;
the third adjacent well comprising a transparent polymer membrane;
[00258] (iii) acquiring a population of T lymphocytes derived from the
potential
donor allogeneic to the recipient subject;
[00259] (iv) seeding and expanding in a fourth adjacent well of the multiwall
plate-based perfusion culture device the population of T lymphocytes of (iii),
[00260] (v) co-culturing in the second liquid medium the population of tumor
cells
comprising one or more tumor-specific antigens that is derived from the
recipient subject in
the third adjacent well and the population of T lymphocytes derived from the
potential donor
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allogeneic to the recipient subject in the fourth adjacent well, the co-
culturing being
characterized by:
[00261] the third adjacent well being fluidly connected to the fourth adjacent
well
so that the second liquid medium is flowable between the third adjacent well
and the fourth
adjacent well;
[00262] an interaction between the population of tumor cells comprising one or
more tumor-specific antigens that is derived from the recipient subject and
the population of
T lymphocytes derived from the potential donor allogeneic to the recipient
subject that is
effective to generate alloreactive effector T lymphocytes derived from the
potential donor;
[00263] (vi) measuring damage to the population of tumor cells derived from
the
recipient subject induced by the alloreactive effector T lymphocytes derived
from the
potential donor allogeneic to the recipient subject, wherein the damage is a
measure of an
effective graft versus tumor (GVT) response against the tumor-specific
antigens; and
[00264] (vii) ranking a plurality of potential donors by the measure of the
effective
GVT response against the tumor-specific antigens; and
[00265] C. Treating the recipient subject with a BMT graft derived from a
selected
donor allogeneic to the recipient, the selected donor being characterized by a
reduced risk of
intestinal graft versus host disease, and an effective GVT response against
the tumor-specific
antigens.
[00266] With respect to step (A), according to some embodiments, the tissue
sample is a biopsy sample. According to some embodiments, the biopsy sample is
a small
biopsy sample of the order of 3 mm in diameter. According to some embodiments,
the
biopsy sample is collected from intestinal tissue. According to some
embodiments, the
biopsy sample is collected from intestinal tissue by colonoscopy, endoscopy,
or a
combination thereof.
[00267] According to some embodiments the potential donor is a haploidentical
donor (i.e., parent, child and other close relative).
[00268] According to some embodiments, the patient sample is acquired soon
after
diagnosis of a hematological malignancy for which allogeneic BMT is a
potential therapeutic
approach and stored for later use in the method. According to some
embodiments, the patient
sample is acquired in the relapse setting after chemotherapeutic interventions
have been
exhausted.
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[00269] According to some embodiments, the primary IECs are prepared from the
small intestine, large intestine, or colon of a recipient subject, and
expanded using conditional
reprogrammed cell (CRC) technology, which comprises cultivating the primary
IECs in a
CRC medium containing ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2
fibroblast
feeder layer. According to some embodiments the medium for cultivating the
primary
human IECs containing ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2
fibroblast
feeder layer is RPMI.
[00270] According to some embodiments, the CRC medium is replaced with
compete RPI-1640 medium (RPMI-1640 supplemented with 10% fetal bovine serum
and 5%
L-glutamine) to culture the T cells.
[00271] According to some embodiments, the first well comprises a nanofibrous
coated transparent polymer membrane. According to some embodiments, the
nanofibrous
coating is prepared by electrospinning. According to some embodiments, the
nanofibrous
coating comprises a fiber matrix of polycaprolactone in which ECM-like
molecules (e.g.,
collagen) is dispersed. According to some embodiments, the nanofibrous coated
transparent
polymer membrane is effective to maintain the long-term functionality of
CRIECs and T cells
using RPMI as a common culture medium.
[00272] According to some embodiments, the method comprises providing
polymer microbeads preconditioned with one or more adhesion-promoting agents
to promote
adhesion of cells to at least one surface of the microbeads. According to some
such
embodiments, the cells are dendritic cells (DCs). According to some
embodiments, the
adhesion promoting agent comprises an effective amount of lipopolysaccharides
(LPS),
wherein the LPS are effective to promote adhesion of the DCs to the microbeads
surface.
[00273] According to some embodiments, the first well of the device contains a
population of conditionally reprogrammed IECs prepared from a mammal, and the
second
well fluidly connected to the first well contains T cells comprising dendritic
cells from an
allogeneic mammal. According to some such embodiments, the mammal is a mouse.
According to some such embodiments, the mammal is a human.
[00274] According to some embodiments, a CRIEC culture chamber can be
established by placing CRIECs into the first well on top of a polymer membrane
coated with
an electrospun PCL/collagen nanofibrous mesh.
[00275] According to some embodiment, an average open space (or pore size) in
the nanofibrous mesh is within a range of about 1-10 p.m. According to some
such
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embodiments, the average open space (or pore size) in the nanofiber mesh is
about 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 p.m.
[00276] According to some embodiments, an antigen presenting cell (APC)
culture
chamber can be established by placing T cells comprising DCs in the liquid
medium onto the
pre-treated polymer microbeads in the second well. According to some
embodiments, one or
more cytokines can be added to the culture chamber to prolong T cell
maintenance.
According to one embodiment, the population of T-cells suspended in the liquid
medium
comprises about 105 to 106 cells.
[00277] According to some embodiments, the method further comprises
replenishing DCs with new DCs by opening a polymer plug on the top of the APC
chamber
and placing new DCs onto the top of the microbead assembly. According to some
embodiments, the dendritic cell assembly can be replaced by a new
microbead/dendritic cell
assembly.
[00278] According to some embodiments, an average size of a polymer microbead
is in a range of about 45-90 p.m. According to some such embodiments, the
average size of a
polymer microbead is about 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 p.m.
[00279] According to some embodiments, the T cells comprising dendritic cells
are
derived from peripheral blood lymphocytes. According to some embodiments,
mouse
dendritic cells are enriched by injecting host mice with a B16-FLt3L tumor.
[00280] According to some embodiments, the polymer membrane has an average
pore size that provides a sufficient opening for T cells to go through.
According to some
such embodiments, the average pore size of the polymer membrane is about 7-13
p.m.
According to some such embodiments, the average pore size of the polymer
membrane is
about 7, 8, 9, 10, 11, 12 or 13 p.m. According to some such embodiments, an
average
diameter of a T cell is about 5 p.m.
[00281] According to some embodiments, the nanofibrous coated transparent
polymer membrane is effective to anchor a population of cells. According to
some
embodiments, the polymer membrane comprises the population of human intestinal
epithelial
cells, the population of CRIECs, or a combination thereof.
[00282] According to some embodiments the cells to be cultured can be cultured
in
free suspensions, encapsulated in suitable hydrogels, encapsulated in
matrices, and/or
encapsulated in scaffolds. For example, according to some embodiments, the T
cells
comprising a suspension of about 106 T cells (e.g., eGFP m-T cells (harvested
from an eGFP
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transgenic B6 mouse) or h-T cells) in a culture medium are flowable, i.e.,
they circulate with
the liquid medium of the microfluidic well plate-based perfusion culture
device. According
to one embodiment, the culture medium contains retinoic acid, which
facilitates the
generation of T cells with superior IEC-killing avidity.
[00283] According to some embodiments, a ratio of CRIECs: T cells is in a
range
of from 2:1 to 20:1. According to some such embodiments, the ratio of CRIECS:
T cells is
2:1, 34:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1,
16:1, 17:1, 18:1, 19:1,
or 20:1.
[00284] According to some embodiments, a ratio of T cells: DCs is in a range
of
from 1:1 to 20:1. According to some such embodiments, the ratio of T cells:
DCs is 1:2, 2:1,
3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12: 1, 13:1, 14:1, 15:1, 16:1,
17:1, 18: 1, 19:1, or
20:1. According to some such embodiments, the ratio of T cells: DCs is 5:1.
[00285] According to some embodiments, the co-culturing of the population of
human IECs from the proposed recipient subject and the population of T
lymphocytes from
the donor allogeneic to the recipient subject is effective to generate
alloreactive
activated/effector T-lymphocytes. According to some embodiments, the
allogeneic
activated/effector T cells recirculate through interconnected h-CRIEC and
antigen presenting
cell (APC) culture chambers. According to some embodiments, the alloreactive
activated/effector T-lymphocytes comprise a population of antigen presenting
cells.
According to some such embodiments, the population of antigen presenting cells
comprises a
population of dendritic cells. According to some embodiments, the alloreactive
T cells
become activated by cognitive alloantigens on h-CRIECs. According to some
embodiments,
the activated alloreactive T cells comprise activated antigen presenting cells
(APCs).
According to some embodiments, the APCs comprise activated/effector dendritic
cells.
According to some embodiments, the alloreactive activated/effector T cells are
effective to
induce quantifiable damage to the population of h-CRIECs. According to some
such
embodiments, the device is clinically viable, i.e., it is effective to
increase the critical number
of functional T cells required to induce quantifiable alloreactivity in the
CRIEC culture
chamber within a diagnostic screening time frame of 2-3 weeks.
[00286] According to some embodiments, quantifiable damage to the population
of
CRIECs comprises measurable killing of the population of CRIECS.
[00287] According to some embodiments, the recipient subject and potential
allogeneic donor are mammals.
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[00288] According to some embodiments, the recipient subject and potential
allogeneic donor are mice. According to some embodiments, the recipient
subject and
potential allogeneic donor are human.
[00289] With respect to step B, according to some embodiments, the sample
procured from the recipient patient with a hematologic malignancy is a blood
sample, a bone
marrow sample, or a leukapheresis sample. According to some embodiments, a
sample is
also procured from a normal, noncancerous subject to serve as a non-tumoral
reference
sample.
[00290] According to some embodiments, the patient sample is acquired soon
after
diagnosis of a hematological malignancy for which allogeneic BMT is a
potential therapeutic
approach and stored for later use in the method. According to some
embodiments, the patient
sample is acquired in the relapse setting after chemotherapeutic interventions
have been
exhausted.
[00291] According to some embodiments, the cell population of interest can be
selected by any techniques known to the skilled artisan. For example, without
limitation,
according to some embodiments, cells expressing a particular cell antigen are
selected by
fluorescence activated cell sorting (FACS). According to some embodiments,
cells
expressing a particular cell antigen are selected by positive or negative
immunoseparation
techniques. According to some embodiments, isolation and/or purification of
cells of interest
from the bone marrow is based on cell fractionation methods based on size and
cell density,
efflux of metabolic dyes, or resistance to cytotoxic agents. According to some
embodiments,
RBCs are depleted by centrifugation. According to some embodiments,
centrifugation at
1000xg for 20 minutes at ambient temperature is performed to separate a thin
grayish white
fraction of a blood sample that contains most of the white blood cells
(leukocytes) (the buffy
coat) from the RBCs.
[00292] According to some embodiments, the tumor samples are cultivated in a
medium containing ROCK inhibitor Y-27632 and an irradiated Swiss 3T3-J2
fibroblast
feeder layer (CRC medium) to generate expanded conditionally reprogrammed
cells.
According to some embodiments, the CRC medium is replaced with compete RPI-
1640
medium (RPMI-1640 supplemented with 10% fetal bovine serum and 5% L-glutamine)
to
culture the T cells.
[00293] According to some embodiments, the third well comprises a nanofibrous
coated transparent polymer membrane. According to some embodiments, the
nanofibrous
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coating is prepared by electrospinning. According to some embodiments, the
nanofibrous
coating comprises a fiber matrix of polycaprolactone in which ECM-like
molecules (e.g.,
collagen) is dispersed. According to some embodiments, the nanofibrous coated
transparent
polymer membrane is effective to maintain the functionality of the tumor cells
and T cells
using RPMI as a common culture medium.
[00294] According to some embodiments, the method comprises providing
polymer microbeads preconditioned with one or more adhesion-promoting agents
to promote
adhesion of cells to at least one surface of the microbeads. According to some
such
embodiments, the cells are dendritic cells (DCs). According to some
embodiments, the
adhesion promoting agent comprises an effective amount of lipopolysaccharides
(LPS),
wherein the LPS are effective to promote adhesion of the DCs to the microbeads
surface.
[00295] According to some embodiments, a tumor cell culture chamber can be
established by placing tumor cells into the third well on top of a polymer
membrane coated
with an electrospun PCL/collagen nanofibrous mesh.
[00296] According to some embodiment, an average open space or pore size in
the
nanofibrous mesh is within a range of about 1-10 p.m. According to some such
embodiments,
the average open space (or pore size) in the nanofiber mesh is about 1, 2, 3,
4, 5, 6, 7, 8, 9, or
p.m.
[00297] According to some embodiments, the antigen presenting cell (APC)
culture chamber can be established by placing T cells comprising DCs in the
liquid medium
onto the pre-treated polymer microbeads in the second well. According to some
embodiments, one or more cytokines can be added to the culture chamber to
prolong T cell
maintenance. According to one embodiment, the population of T-cells suspended
in the
liquid medium comprises about 105 to 106 cells.
[00298] According to some embodiments, the method further comprises
replenishing DCs with new DCs by opening a polymer plug on the top of the APC
chamber
and placing new DCs onto the top of the microbead assembly. According to some
embodiments, the dendritic cell assembly can be replaced by a new
microbead/dendritic cell
assembly.
[00299] According to some embodiments, an average size of a polymer microbead
is in a range of about 45-90 p.m. According to some such embodiments, the
average size of a
polymer microbead is about 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 p.m.
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[00300] According to some embodiments, the T cells comprising dendritic cells
are
derived from peripheral blood lymphocytes. According to some embodiments,
mouse
dendritic cells are enriched by injecting host mice with a B16-FLt3L tumor.
[00301] According to some embodiments, the polymer membrane has an
average
pore size that provides a sufficient opening for T cells to go through.
According to some
such embodiments, the average pore size of the polymer membrane is about 7-13
p.m.
According to some such embodiments, the average pore size of the polymer
membrane is
about 7, 8, 9, 10, 11, 12 or 13 p.m. According to some such embodiments, an
average
diameter of a T cell is about 5 p.m.
[00302] According to some embodiments, the nanofibrous coated transparent
polymer membrane is effective to anchor a population of cells. According to
some
embodiments the cells to be cultured can be cultured in free suspensions,
encapsulated in
suitable hydrogels, encapsulated in matrices, and/or encapsulated in
scaffolds. For example,
according to some embodiments, the T cells comprising a suspension of about
106 T cells
(e.g., eGFP m-T cells (harvested from an eGFP transgenic B6 mouse) or h-T
cells) in a
culture medium are flowable, i.e., they circulate with the liquid medium of
the microfluidic
well plate-based perfusion culture device.
[00303] According to some embodiments, a ratio of tumor cells: T cells is in a
range of from 2:1 to 20:1. According to some such embodiments, the ratio of
CRIECS: T
cells is 2:1, 34:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1,
14:1, 15:1, 16:1, 17:1,
18:1, 19:1, or 20:1.
[00304] According to some embodiments, a ratio of T cells: DCs is in a range
of
from 1:1 to 20:1. According to some such embodiments, the ratio of T cells:
DCs is 1:2, 2:1,
3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12: 1, 13:1, 14:1, 15:1, 16:1,
17:1, 18: 1, 19:1, or
20:1. According to some such embodiments, the ratio of T cells: DCs is 5:1.
[00305] According to some embodiments, the co-culturing of the population of
tumor cells from the recipient subject and the population of T lymphocytes
from the donor
allogeneic to the recipient subject is effective to generate alloreactive
activated T-
lymphocytes. According to some embodiments, the allogeneic activated/effector
T cells
recirculate through the tumor cell culture chamber and the antigen presenting
cell (APC)
culture chamber, wherein the chambers are interconnected. According to some
embodiments, the alloreactive activated T-lymphocytes comprise a population of
antigen
presenting cells. According to some such embodiments, the population of
antigen presenting
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cells comprises a population of dendritic cells. According to some
embodiments, cognitive
alloantigens on the tumor cells are effective to activate the alloreactive T
cells. According to
some embodiments, the activated alloreactive T cells comprise activated
antigen presenting
cells (APCs). According to some embodiments, the APCs comprise
activated/effector
dendritic cells. According to some embodiments, the alloreactive
activated/effector T cells
are effective to induce quantifiable damage to the population of tumor cells,
compared to a
normal control. According to some such embodiments, the device is clinically
viable, i.e., it
is effective to increase the critical number of functional T cells required to
induce
quantifiable alloreactivity in the tumor cell culture chamber within a
diagnostic screening
time frame of 2-3 weeks.
[00306] According to some embodiments, quantifiable damage to the expanded
population of tumor cells comprises measurable killing of the expanded
population of tumor
cells.
[00307] According to some such embodiments, a panel of cell death analysis
methods is used to quantify tumor cell death. For example, annexin V+/PI+
staining using
flow cytometry and in situ detection of cleaved caspase-3 using
immunofluorescence can be
used to determine cell death. According to some embodiments, the percentage of
dead cells
is calculated as [% of Annexin V+/PI+ cells in co-cultures - % of Annexin
V+/PI+ cells in
tumor cells alone cultures] (flow cytometric measurement). According to some
embodiments, CD3 staining is performed to identify adherent T cells
contributing to the
response. According to some embodiments, the caspases-3 staining is conducted
in the well
plate to determine cell death as [fluorescence intensity in co-culture -
fluorescence intensity
in tumor cells alone cultures]/[fluorescence intensity of DAPI staining, as an
indicator of the
number of nucleated cells in the cultures].
[00308] Where a range of values is provided, it is understood that each
intervening
value, to the tenth of the unit of the lower limit unless the context clearly
dictates otherwise,
between the upper and lower limit of that range and any other stated or
intervening value in
that stated range is encompassed within the invention. The upper and lower
limits of these
smaller ranges which may independently be included in the smaller ranges is
also
encompassed within the invention, subject to any specifically excluded limit
in the stated
range. Where the stated range includes one or both of the limits, ranges
excluding either both
of those included limits are also included in the invention.
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[00309] Unless defined otherwise, all technical and scientific terms used
herein
have the same meaning as commonly understood by one of ordinary skill in the
art to which
this invention belongs. Although any methods and materials similar or
equivalent to those
described herein can also be used in the practice or testing of the described
invention,
exemplary methods and materials have been described. All publications
mentioned herein
are incorporated herein by reference to disclose and described the methods
and/or materials
in connection with which the publications are cited.
[00310] It must be noted that as used herein and in the appended claims, the
singular forms "a", "and", and "the" include plural references unless the
context clearly
dictates otherwise.
[00311] The publications discussed herein are provided solely for their
disclosure
prior to the filing date of the present application and each is incorporated
by reference in its
entirety. Nothing herein is to be construed as an admission that the described
invention is not
entitled to antedate such publication by virtue of prior invention. Further,
the dates of
publication provided may be different from the actual publication dates which
may need to be
independently confirmed.
EXAMPLES
[00312] The following examples are put forth so as to provide those of
ordinary
skill in the art with a complete disclosure and description of how to make and
use the present
invention, and are not intended to limit the scope of what the inventors
regard as their
invention nor are they intended to represent that the experiments below are
all or the only
experiments performed. Efforts have been made to ensure accuracy with respect
to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors and
deviations should be
accounted for. Unless indicated otherwise, parts are parts by weight,
molecular weight is
weight average molecular weight, temperature is in degrees Centigrade, and
pressure is at or
near atmospheric.
Example I
[00313] Generating conditionally reprogrammed IECs from murine samples.
Successful isolation of primary murine m-IECs (small intestine) from adult 6-8
week old
mice was performed as detailed by Evans et al. ("The development of a method
for the
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preparation of rat intestinal epithelial cell primary cultures," J. Cell Sci.
101 ( Pt 1), 219-231
(1992)), with modifications from techniques in Zilberberg's lab.
[00314] Expansion of m-IECs was performed using CR (Palechor-Ceron, N. et al.,
"Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK
inhibitor to
conditionally reprogram and immortalize epithelial cells," Am. J. Pathol. 183,
1862-1870,
doi:10.1016/j.ajpath.2013.08.009 (2013); Liu, X. et al., "ROCK inhibitor and
feeder cells
induce the conditional reprogramming of epithelial cells," Am. J. Pathol 180,
599-607, doi:
10.1016/j.ajpath.2011.10.036 (2012); Suprynowicz, F. A. et al., "Conditionally
reprogrammed cells represent a stem-like state of adult epithelial cells,"
Proc. Nat. Acad.
Sci. USA 109, 20035- 20040, doi: 10.1073/pnas. 1213241109 (2012)). Freshly
isolated m-
IECs and m-CRIEC were >98% positive pan cytokeratin and epithelial cell
adhesion
molecule (EpCAM) (ref. 14, 40) positive, confirming the purity of our cultures
(see Fig. 2a
and 2b). Gene expression analysis also corroborated that these cells
significantly expressed
cytokeratin 8 (KRT8; a specific marker for IECs) with low expression of
cytokeratin 15
(KRT15; a marker for skin epithelium (Zhan, Q. et al., "Cytokeratin 15-
positive basal
epithelial cells targeted in graft-versus-host disease express a constitutive
antiapoptotic
phenotype, J. Invest. Dermatol. 127, 106-115, doi:10.1038/sj.jid.5700583
(2007); Whitaker-
Menezes, D., et al, "An epithelial target site in experimental graft-versus-
host disease and
cytokine-mediated cytotoxicity is defined by cytokeratin 15 expression,"
Biology of Blood
and Marrow Transplantation: 9, 559-570 (2003).) (data not shown).
[00315] Upon CR expansion with conditioned medium (Palechor-Ceron, N. et al.,
"Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK
inhibitor to
conditionally reprogram and immortalize epithelial cells," Am. J. Pathol. 183,
1862-1870,
doi:10.1016/j.ajpath.2013.08.009 (2013).), mCRIECs acquired a stem-like
phenotype
(increased CD24 and Lgr5 in the case of IECs) as reported to be the case with
other primary
epithelial cells (Saenz, F. R. et al., "Conditionally reprogrammed normal and
transformed
mouse mammary epithelial cells display a progenitor-cell-like phenotype," PloS
One 9,
e97666, doi:10.1371/joumal.pone.0097666 (2014); (2014); Suprynowicz, F. A. et
al.,
"Conditionally reprogrammed cells represent a stem-like state of adult
epithelial cells," Proc.
Nat. Acad. Sci. USA 109, 20035- 20040, doi: 10.1073/pnas. 1213241109 (2012))
undergoing
CR (Fig. 2b). These results with m-IECs suggest that CRIECs can be produced
while
preserving characteristic IEC functions.
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Example 2
Collecting human biospecimens:
[00316] Donor and recipient peripheral blood lymphocytes (PBL) and allogeneic-
BMT recipient GI biopsies (e.g., taken from colonoscopies) will be collected
in accordance
with the IRB approved protocol. GI specimens will be collected at the onset of
GVHD if GI
biopsies already are being performed. Transplant patients, undergoing a gut
biopsy as part of
their standard of care, will be asked to donate two extra cores of
approximately 3 mm in size.
Overly inflamed tissue samples will not be used in this study.
Blood collection will be performed prior to transplant to ensure the
collection of viable cells
(four 8.5 mL yellow top tubes per individual, containing 106 cells/mL, of
which half are T
cells). Donor blood will be used to isolate T cells for killing assays, and
patient blood will be
utilized to develop DCs as specified below. PBL will be obtained by
centrifugation of blood
samples over Ficoll-Paque-Plus (Friedman, T. M. et al., "Overlap between in
vitro donor
antihost and in vivo posttransplantation TCR Vbeta use: a new paradigm for
designer
allogeneic blood and marrow transplantation," Blood 112, 3517- 3525, doi:
10.1182/blood-
2008-03-145391 (2008)) and cryopreserved for later use in killing assays. Upon
collection,
tissue samples will be place in PBS at 4 C.
Example 3
Generating h-CRIECs from patients and murine models:
[00317] h-CRIECs will be prepared following procedures developed for the
generation of m-CRIECs (see, e.g., Saenz, F. R. et al. Conditionally
reprogrammed normal
and transformed mouse mammary epithelial cells display a progenitor-cell-like
phenotype.
PloS One 9, e97666, doi:10.1371/joumal.pone.0097666; (2014); Palechor-Ceron,
N. et al.
Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK
inhibitor to
conditionally reprogram and immortalize epithelial cells. Am. J. Pathol. 183,
1862-1870,
doi:10.1016/j.ajpath.2013.08.009 (2013); Liu, X. et al. ROCK inhibitor and
feeder cells
induce the conditional reprogramming of epithelial cells, Am. J. Pathol. 180,
599-607, doi:
10.1016/j.ajpath.2011.10.036 (2012)). All CRC will be cryopreserved until use.
Example 4
Validating the utility of the in vitro GVHD (iGVHD) platform using clinically
relevant
murine models of allo-BMT:
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[00318] Table 1. Murine models and experiments for biological validation of
the
iGVHD concept and utility.
Murine BMT model Expected Outcome
miHA model Using the miHA model B6¨>BALB.B, with known GVHD
B6¨>BALB.B potential (Zilberberg, J., McElhaugh, D., Gichuru, L.
N.,
Korngold, R. & Friedman, T. M. Inter-strain tissue-
infiltrating T cell responses to minor histocompatibility
antigens involved in graft-versus-host disease as determined by
Vbeta spectratype analysis, J. Immuno1.180, 5352-5359
(2008)), the percentage of killed IEC to aid the development of
an empirical pathological index (PIdx). This PIdx will be put
in practice to assess donor-patient pair reactivity in clinical
samples.
Negative Control B6¨>CXB-7 will be used as a negative control to
identify the
lower limit of the killing assay, i.e., to help determine what
B6¨>CXB-7 degree of IEC apoptosis can be expected in the absence
of in
vivo GVHD-induced lethality.. No substantial damage of
CXB-7 IEC in this nonlethal miHA model, which has a subset
of the miHA expressed by the BALB-B strain, is expected.
Some apopotosis may occur, since some cachexia can be
observed in recipient mice. (Korngold, R. & Wettstein, P. J.
Immunodominance in the graft-vs-host disease T cell response
to minor histocompatibility antigens. J. Immunol. 145, 4079-
4088 (1990)).
Haploidentical transplant To recapitulate the clinical scenario where
haploidentical
model, with three different transplant recipients undergo cyclophosphamide
treatment on
potential donors and day 3 post-BMT to eliminate highly alloreactive MHC
specific
syngeneic negative control: T cells and thereby lessen the severity of GVHD
(Kanda, J.,
B6¨> B6D2F1; BALB.B Chao, N. J. & Rizzieri, D. A. Haploidentical
transplantation for
¨B6D2F1; C3H. SW¨> leukemia, Cur. Oncol. Reports 12, 292-301, doi:
10.1007/s1
B6D2F1; B6D2F1 ¨> 1912-010-0113-4 (2010);
Luznik, L., O'Donnell, P. V. &
B6D2F1. Fuchs, E. J. Post-transplantation cyclophosphamide for
tolerance induction in HLA-haploidentical bone marrow
transplantation. Sem. Oncol. 39, 683-693, doi:
10.1053/j.seminonco1.2012.09.005 (2012)), cultures will also
be treated with an analog of cyclophosphamide as described
(Kanakry, C. G. et al. Aldehyde dehydrogenase expression
drives human regulatory T cell resistance to posttransplantation
cyclophosphamide. Sci. Translat. Med. 5, 21 Ira 157, doi:
10.1126/scitranslmed.3006960 (2013)). This would leave the T
cell responses to be directed mostly to miHA differences.
The killing assays will thus be utilized here to predict the best
donor for the B6D2F1 recipient, i.e., the donor that will incur
the least degree of pathological damage (as determined by the
PIdx). It is expected that the donor with the lowest PIdx score
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Murine BMT model Expected Outcome
will likely induce less GVHD in vivo. This will be correlated
with in vivo BMT GVHD experiments using methodology that
has been described (Fanning, S. L. et al Unraveling graft-
versus-host disease and graft-versus-leukemia responses
using TCR Vbeta spectratype analysis in a murine bone
marrow transplantation model. J. Immunol. 190,447-457, doi:
10.4049/jimmunol. 1201641 (2013); Zilberberg, J., et al., Inter-
strain tissue-infiltrating T cell responses to minor
histocompatibility antigens involved in graft-versus-host
disease as determined by Vbeta spectratype analysis. J.
Immunol 180, 5352-5359 (2008)). The syngeneic negative
control using B6D2F1 donor cells will provide the baseline for
the PIdx.
[00319] Preliminary results suitability of cRPMI: Since CRC medium additives
(e.g., ROCK kinase inhibitor) can ameliorate GVHD (Iyengar, S., Zhan, C., Lu,
J., Korngold,
R. & Schwartz, D. H. Treatment with a Rho Kinase Inhibitor Improves Survival
from Graft-
Versus-Host Disease in Mice after MHC-Haploidentical Hematopoietic Cell
Transplantation.
Biol. Blood Marrow Transplant.:, doi:10.1016/j.bbmt.2014.04.029 (2014)) and
therefore
should not be used for co-culture of m-CRIECs and T cells, the CRC medium was
replaced
with complete RPMI-1640 medium (cRPMI, RPMI medium supplemented with 10% FBS
and 5% L-glutamine), which is conventionally used to culture T cells
(Friedman, T. M. et al.
Overlap between in vitro donor antihost and in vivo posttransplantation TCR
Vbeta use: a
new paradigm for designer allogeneic blood and marrow transplantation. Blood
112, 3517-
3525, doi: 10.1182/blood-2008-03-145391 (2008)) Fig. 2b. m-CRIEC's
upregulation of
surface expression of major histocompatibility complexes I and II (MHCI and
MHC II; the
murine equivalent of human HLA) (Fig. 3). The increased expression of these
molecules
serves as a catalytic step without which T cells cannot recognize miHA or any
antigen on the
surface of host cells (Korngold, R. & Sprent, J. Graft-versus-host disease in
experimental
allogeneic bone marrow transplantation. Proc. Soc. Exp. Biol. Med. Soc. Exp.
Biol. Med.
197, 12-18 (1991); Korngold, R. & Sprent, J. Surface markers of T cells
causing lethal graft-
vs-host disease to class I vs class II H-2 differences; J. Immunol. 135, 3004-
3010 (1985)).
The results indicate that, while other culture media partially hindered the
upregulation of
these molecules, cRPMI permitted maximum expression of MHC-I and MHC-II after
72 h of
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cytokine exposure.
Example 5
Feasibility of using nanofibrous mesh in maintaining the long-term
functionality of
CRIECS:
[00320] Basement membrane (BMa)-like fibrous meshes with random fiber
organization were prepared by electrospinning (Yang, X., Ogbolu, K. R. & Wang,
H.
Multifunctional Nanofibrous Scaffold for Tissue Engineering. J. Exp.
Nanoscience 3, 329 -
345 (2008)). To obtain stable and strong nanofibers, slow degradable,
biocompatible
polycaprolactone (PCL) was used as the fiber matrix phase in which Type IV
collagen
(representing ECM molecules) was dispersed.
[00321] As shown in Fig. 4, the combination of nanofibrous mesh and cRPMI
culture medium enabled the maintenance of viable and morphologically sound
CRIECs, even
after 7 days in the absence of CR medium.
[00322] As shown in Table 2 flow cytometric analysis of annexin V+/propidium
iodide (PI) + staining showed that mCRIEC viability decreased at an E:T ratio
of 5:1 as
determined by increased apoptotic cells (% Annexin V+). At a ratio of 10:1,
64.8% of m-
CRIEC were dead (double+) by day 6. PIdx=3.83.
[00323] Table 2:
E:T ratio % Annexin V+ % AnV+/PI+
m-CRIEC 14.5 11.8
5:1 71.3 19.3
10:1 67.4 64.8
[00324] Also, the above culture conditions were sufficient to enable anti-
allogeneic
T cell responses capable of inducing quantifiable reaction to m-CRIECs in an
MHC-
mismatched setting (Fig. 5a, and Table 1). The pathological index (PIdx) was
calculated to
be 3.83.
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Example 6
Experiments to establish the pathological index (PIdx) to quantify T cell
induced IEC
damage and killing:
[00325] The predictive capability of co-culture killing assays and later iGVHD
will
be compared to known in vivo outcomes from well-established murine models of
BMT(Table
1). These models represent different degrees of alloantigenic barriers and
hence distinct
clinical scenarios:
[00326] the miHA-disparate C57BL65 (B6) ¨> C.B10-H2b/LiMedJ (BALB.B) and
B6 ¨> CXB-7/By (CXB-7) models (see Zilberberg, J. et al, "Inter-strain tissue-
infiltrating T
cell responses to minor histocompatibility antigens involved in graft-versus-
host disease as
determined by Vbeta spectratype analysis," J. Immunol. 180: 5352-59 (2008);
Korngold, R.
& Wettstein, P.J. "Immunodominance in the graft vs host disease T cell
response to minor
histocompatibility antigens," J. Immunol. 145: 4079-4088 (1990); Jones, S.C.
et al, "Specific
donor Vbeta-associated CD4 T-cell responses correlate with severe acute graft
versus host
disease directed to multiple minor histocompatibility antigens. Biol.Blood
Marrow
Transplant. 10: 91-105, doi: 10.1016/j.bbmt.2003.10.002 (2004); Jones et al,
"Importance
of minor histocompatibility antigen expression by nonhematopoietic tissues in
a CD4+ T
cell-mediated graft-versus-host disease model," J. Clin. Invest. 112: 1880-86,
doi:
10.1172/JC119427 (2003); Friedman, T.M., et al, "Vbeta spectratype analysis
reveals
heterogeneity of CD4+ T cell responses to minor histocompatibility antigens
involved in
graft-versus-host disease: correlations with epithelial tissue infiltrate,"
Biol. Blood Marrow
Transplant. 7: 2-13, doi: 10.1053/bbmt.2001.v7.pm11215694 (2001); Friedman,
T.M. et al,
"Repertoire analysis of CD8+ T cell responses to minor histocompatibility
antigens involved
in graft-versus-host disease, J. Immunol 161: 41-48 (1998)), where both donor
and recipients
are MHC (H2b ¨ matched); and
[00327] the haploidentical-MHC model (see Zilberberg, J. et al, "Inter-strain
tissue-infiltrating T cell responses to minor histocompatibility antigens
involved in graft-
verus-host disease as determined by Vbeta spectratype analysis," J. Immunol.
180: 5352-59
(2008); Korngold, R. & Wettsstein, P.J. "immunodominance in the graft vs host
disease T
cell response to mino histocompatibility antigens," J. Immunol. 145: 4079-4088
(1990);
Jones, S.C. et al, "Specific donor Vbeta-associated CD4 T-cell responses
correlate with
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severe acute graft versus host disease directed to multiple minor
histocompatibility antigens.
Biol. Blood Marrow Transplant.10: 91-105, doi: 10.1016/j.bbmt.2003.10.002
(2004); Jones
et al, "Importance of minor histocompatibility antigen expression by
nonhematopoietic
tissues in a CD4+ T cell-mediated graft-versus-host disease model," J. Clin.
Invest. 112:
1880-86, doi: 10.1172/JC119427 (2003); Friedman, T.M., et al, "Vbeta
spectratype analysis
reveals heterogeneity of CD4+ T cell responses to minor histocompatibility
antigens involved
in graft-versus-host disease: correlations with epithelial tissue infiltrate,"
Biol.Blood Marrow
Transplant.7: 2-13, doi: 10.1053/bbmt.2001.v7.pm11215694 (2001); Friedman,
T.M. et al,
"Repertoire analysis of CD8+ T cell responses to minor histocompatibility
antigens involved
in graft-versus-host disease, J. Immunol 161: 41-48 (1998)) B6 ¨>
(B6xDBA/2)F1[B6D2F1(H2b/d)] (Patterson, A.E. and Korngold, R., "Infusion of
select
leukemia-reactive TCR Vbeta+ T cells provides graft-versus-leukemia responses
with
minimization of graft-versus-host disease following murine hematopoietic stem
cell
transplantation," Biol.Blood Marrow Transplant.7: 187-196 (2001)). In brief, m-
IECs from
small and large intestine can be isolated from recipient strains and expanded
using CR
technology. The m-CRIECs can be cryopreserved for later use in co-culture
experiments.
The m-CRIECs can be cultured on nanofibrous matrices in the presence of
complete RPMI
supplemented with TNF-a and IFN-y to induce upregulation of MHC-1 and MHC-II
molecules.
[00328] In brief, for each of the experimental murine models proposed in Table
1,
m-IECs (from small and large intestine) will be isolated from recipient
strains and expanded
using CR technology. The m-CRIECs will be cryopreserved for later use in co-
culture
experiments. The m-CRIECs will be cultured on nanofibrous matrices in the
presence of
cRPMI supplemented with TNFa and IFNy to induce upregulation of MHC-1 and MHC-
II
molecules; an indispensable state to generate tissue-directed alloresponses
(Fig. 3).
[00329] Although TNFa is best known for its inflammatory effects, it also can
induce upregulation of programmed death ligand 1 (PDL-1) on the surface of
cells, which
acts as an immunological checkpoint and can shut down effector T cells.
Preliminary data
(not shown) indicates that epithelial cells upregulate PDL-1 under
inflammatory conditions
(Wu, Y. Y. et al Increased programmed death-ligand-1 expression in human
gastric epithelial
cells in Helicobacter pylori infection. Clin. Exp. Immunol. 161, 551-559, doi:
10.1111/j.
1365- 2249.2010.04217.x (2010)), and thus TNFa can play an important
regulatory role in
allogeneic transplantation (Alderson, K. L. et al Regulatory and conventional
CD4+ T cells
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show differential effects correlating with PD-1 and B7-H1 expression after
immunotherapy.
J. Immunol. 180, 2981-2988 (2008); Tanaka, K. et al PDL1 is required for
peripheral
transplantation tolerance and protection from chronic allograft rejection. J.
Immunol. 179,
5204-5210 (2007); Saha, A. et al Host programmed death ligand 1 is dominant
over
programmed death ligand 2 expression in regulating graft-versus-host disease
lethality. Blood
122, 3062-3073, doi: 10.1182/blood-2013-05- 500801 (2013)). A PDL-1 blocker
(e.g.,
MPDL3280A, Genentech) , will be introduced in order to ensure that T cell
reactivity is not
negatively modulated through this pathway.
[00330] Likewise, to better recapitulate tissue-induced damage by
preconditioning
regimens (Ferrara, J. L., Levine, J. E., Reddy, P. & Holler, E. Graft-versus-
host disease.
Lancet 373, 1550-1561, doi: 10.1016/S0 140-6736(09)60237-3 (2009)), IEC can be
treated
with the same chemotherapeutic agents that patients typically receive prior to
transplant.
This may induce the expression of MHC-I and MHC-II on the IEC, priming the T
cells for a
more robust response.
Mixed Lymphocyte Culture
[00331] To mimic the early activation/proliferation stage of T cells in the
described
in vitro system, a mixed lymphocyte culture (MLC) will be used. (Fanning, S.
L. et al
Unraveling graft-versus-host disease and graft-versus-leukemia responses using
TCR Vbeta
spectratype analysis in a murine bone marrow transplantation model. J.
Immunol. 190,447-
457, doi: 10.4049/jimmunol. 1201641 (2013); Friedman, T. M. et al. Overlap
between in
vitro donor antihost and in vivo posttransplantation TCR Vbeta use: a new
paradigm for
designer allogeneic blood and marrow transplantation. Blood 112, 3517- 3525,
doi:
10.1182/blood-2008-03-145391 (2008)). In brief, donor T cells (i.e.,
responders; R) will be
cultured with irradiated (30 Gy) recipient lymphocytes (i.e., stimulators; S)
at a 1:2 R:S ratio.
For human MLC, enriched PBL from the patients will be used to stimulate
responding T cells
from their donors. Natural killer cells will be depleted from donor T cells to
diminish non-
specific target cell killing by this subpopulation of lymphocytes. After 9
days, human MLC
responders will be harvested and re-stimulated for another 8 days as before,
with the addition
of 20 U/ml of rIL-2.
MLC will be carried out in the antigen presenting cells (APC) culture chamber,
as part of the
iGVHD platform, to facilitate activation, expansion and concentration of
alloreactive T cells.
Dendritic cells, as opposed to bulk lymphocytes, will be used in iGVHD, with a
T cell-DC
(R:S) ratio of 5:1. Activated T cells from MLC will then be placed in CRIEC on
nanofibers
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to monitor for epithelial cell death. Killing assays with specimens from
murine models of
allo-BMT (Table 1), where the GVHD response has been characterized in vivo,
will be used
in order to designate an empirical PIdx to quantitate the response.
Objective Cell Death Analysis Methods
[00332] A panel of objective cell death analysis methods (e.g., Annexin V/PI
staining) using flow cytometry and in situ detection of cleaved caspase-3
using
immunofluorescence will be utilized to determine cell death. The percentage of
dead cells is
calculated as [% of Annexin V+/PI+ cells in co-cultures - % of Annexin V+/PI+
cells in IEC
alone cultures] (flow cytometric measurement). CD3 staining also will be
performed to
identify adherent T cells contributing to the response.
[00333] Caspase-3 staining also will be conducted in the well plate (and later
in the
microfluidic chambers to determine cell death as [fluorescence intensity in co-
culture -
fluorescence intensity in IEC alone cultures]/[fluorescence intensity of DAPI
staining, as an
indicator of the number of nucleated cells in the cultures].
[00334] Cell death will be evaluated at three or more (if determined to be
necessary) T cell-IEC ratios (i.e., the effector: target, or E:T ratio).
According to some
embodiments, the E:T ratio is 30, 10, or 3. (See Choksi, S. et al, "A cD8 DE
loop peptide
analog prevents graft versus host disease in a multiple minor
histocompatibility antigen-
mismatched bone marrow transplantation model," Biol.Blood Marrow
Transplant.10: 669-
680, doi: 10.1016/j.bbmt.2004.06.005 (2004)).
[00335] The PIdx will be determined as the slope of the curve of percentage of
dead cells vs. E:T ratios, where a steeper slope indicates a higher risk for
developing GVHD.
The PIdx will be determined at 4 different time points (day 3, day 7, day 14
and day 21 post
co-culture) in order to maximize the opportunity to observe a response while
ensuring that
faster reactions do not reach plateau before obtaining a quantifiable PIdx,
and that slow-to-
develop GVHD responses also can be captured.
[00336] Statistical considerations. Continuous random variables (i.e.,
flow
cytometric data, in situ staining/caspase-3 readout, PIdx) will be summarized
as mean
(standard deviation) or median (interquartile range) depending on whether or
not they are
normally distributed. Categorical random variables (i.e., GVHD grading) will
be presented
as count (percentage). Comparison of continuous random variables between
groups (i.e.,
comparing different murine allo-BMT models) will be performed using two-sided
Student's
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t-test or 2-sided Wilcoxon rank sum test, analysis of variance (ANOVA),
Kruskal-Wallis, as
appropriate. Categorical variables will be compared using Fisher's exact test
or Pearson's
Chi-square test, as appropriate. Median survival of transplanted mice will be
estimated by
the Kaplan-Meier method. Any p<0.05 will be considered statistically
significant. For
reproducibility of PIdx and staining methods, repeated (test-retest)
measurements of PIdx will
be compared using two-sided paired t-tests or Wilcoxon signed rank test.
Correlation of the
replicate PIdx measurements will be examined using Pearson correlation
coefficient or
Spearman correlation coefficients. Reliability of the PIdx will be evaluated
using intra-class
correlation coefficient, coefficient of variation. To examine the effect of
culture time on
PIdx, a mixed model repeated measures analysis will be conducted with PIdx at
different
time points. The Bland-Altman plot will be used to assess agreement between
flow
cytometry and in situ staining.
Example 7
Use of the Multiwell Plate-Based Microfluidic Perfusion Culture Device to
mimic
interactions of circulating murine T cells (m-T cells) with m-CRIECs and
murine dendritic
cells (m-DCs):
[00337] Our current prototype device (Fig. 6) was used to mimic interactions
of
circulating murine T cells (m-T cells) with m-CRIECs and murine dendritic
cells (m-DCs) as
illustrated in Fig. 1. One practical design feature of the device is the use
of removable
polydimethylsiloxane (PDMS) plugs at the top of the culture chambers to: (1)
allow the
convenient placement of cells and biomaterials into the culture chambers at
various time
points during culture; (2) externally interconnect culture the m-CRIEC and m-
DC culture
chambers using polyethylene tubing and a peristaltic pump (Model 78023-02,
ISMATEC);
and (3) recirculate m-T cells suspended in the RPMI common culture medium. The
device
uses transparent polycarbonate (PC) membranes (TCTP02500, Millipore) to: (1)
anchor
tissue cells and biomaterials, and (2) provide optical access through the
bottom of the
chambers for cell characterization with plate readers.
[00338] As shown in Fig. 6c, the prototype device was assembled with: (1) a
commercial polystyrene (PS) bottomless 96-well plate (Model 655-000, Greiner
Bio-One); 2)
three micropatterned PDMS layers made by soft lithography, and (3) one blank
glass layer.
The PDMS layers were used to: (1) provide a microfluidic channel of 200 p.m
thick and 5
mm wide for use as an internal fluidic passage between the chambers, and (2)
anchor the
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placement of the PC membranes within the culture chambers. The bottom of the
device was
sealed with the 1.2 mm-thick glass layer for use with plate readers. These
parts were bonded
using oxygen plasma treatments.
[00339] For our preliminary study, the average pore of the PC membranes was
selected to be 10 i.t.M in order to provide sufficient opening for m-T cells
to go through, since
the average diameter of m-T cells is about 5 p.m. For the m-CRIEC culture
chamber, the
membrane was coated with electrospun PCT/collagen nanofibrous meshes (Fig.
6e).
[00340] The device was used to culture m-CRIECs prepared from the small
intestine of a B6 mouse. The cells develop a confluent layer while maintaining
their viability
up to 7 days (Fig. 7a).
[00341] After the m-CRIEC culture was established, eGFP m-T cells harvested
from an eGFP transgenic B6 mouse were suspended in the culture medium (106
cells total)
and introduced and circulated through the device. The SEM and fluorescence
images in Figs.
7b and 7c show that T cells were able to travel through the PC membrane and
the nanofibrous
meshes and interact with m-CRIECs through physical contact. Accordingly, these
results
show that the device can be used to promote physical interactions between m-
CRIECs and
circulating m-T cells. The a biomaterial that has been commonly used for 3D
lEC/organoid
cultures (Sato, T. et al. Long-term expansion of epithelial organoids from
human colon,
adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterol. 141, 1762-
1772,
doi:10.1053/j.gastro.2011.07.050 (2011)).
[00342] For the m-APC culture chamber, PS microbeads of 90 pm were assembled
with m-DCs (from BALB.B mice) to form a 250 pm-thick assembly on the PC
membrane
surface. Microbeads were pre-conditioned with 100 ng/mL lipopolysaccharides
(LPS) to
promote the adhesion of m-DCs to the microbead surface (Abdi, K., Singh, N. J.
&
Matzinger, P. Lipopolysaccharide-activated dendritic cells: "exhausted" or
alert and waiting?
J.Immunol. 188, 5981-5989, doi: 10.4049/jimmuno1.1 102868 (2012)). m-T cells
(from eGFP
transgenic B6 mice) were labeled with eFIuro 670 and introduced 24 h later
from the top of
the microbeads assembly at a R:S of 5:1. Since the packed 90 pm microbeads
provide
interstitial openings of -14 pm, T cells were able to infiltrate through the
microbeads
assembly and interact with m-DCs, which were attached to the microbead
surface. The m-T
cells were circulated for 4 days. As shown in Fig. 8, analyses of live cells
(as determined by
light forward and side scatters) the P.:S ratio of m-T cells to m-DCs: (1)
increased from 5:1 to
-10:1 (i.e., m-T cells=90%, m-DCs=9%) in the 3D culture chamber and (2)
decreased to -2:1
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(i.e., m-T cells=60%, m-DCs=30%) in 2D plate culture. Likewise, while T cells
underwent
one round of proliferation in both culture conditions (for explanation of
analyses, see Zhang
et al. Patient-Specific 3D Microfluidic Tissue Model for Multiple Myeloma.
Tissue
engineering. Part C, Methods 20: 663-670, doi: 10.1089/ten.TEC.2013.0490
(2014)), the
percentage of proliferating cells (labeled as "P" in Fig. 8) was greater in 3D
than in 2D (40
vs. 33%).
[00343] As hypothesized, these results suggest that the circulatory 3D
perfusion
culture is an effective approach in enhancing the viability, proliferation,
and activation of T
cells in comparison to conventional 2D co-culture. These enhancements are
attributed to the
synergistic use of both microbeads and circulatory perfusion in providing m-T
cells with
significantly higher chances of interacting with m-DCs.
[00344] Taken together, these preliminary results strongly support that the
device
of the described invention can be used for: (1) biomaterials-guided cultures
of CRIECs and
DCs and (2) T cell circulation through these chambers to facilitate and
enhance the viability,
proliferation, and activation of reactive T cell population.
Example 8
Experiments to further optimize the use of the device in replicating the
stimulation,
circulation and proliferation events that donor T cells encounter in the
patient body and
predicting the pathologic potential of donor T cells against host epithelium:
[00345] Experiments to establish that 80% of unstimulated T cells can be
circulated through the CRIEC and DC culture chambers for up to I week. The
effects of
biomaterials, flow conditions, and tissue cell presence on the re-circulation
of unstimulated T
cells from transgenic eGFP B6 mice, in the range of 105 to 106 cells will be
quantified. These
baseline experiments will be primarily conducted with murine cells, but main
results from the
experiments will be confirmed using human cells. When tissue cells are not
present in the
device, it is anticipated that culture medium flow rate, pore size of
nanofibrous mesh, and
microbead size will have major influences as to how T cells can travel through
the culture
chambers. (1) The flow rate will be varied in the range of 10 to 50 uL/min;
(2) the mesh pore
size will be varied from 5 to 10 inn by controlling electrospinning process
parameters, and (3)
the PS microbead size will be varied, i.e., 45, 75, and 90 urn as these sizes
are commercially
available (Polyscience). In addition to qualitative visual and microscopic
observations at
various locations of the device, the percentage changes of circulating T cells
(vs. cells that get
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entrapped in the device) will be quantified by sampling 50-100 uL of the
effluent each day
for a 1 week period and counting the cells in the collected medium using an
automated cell
counter. Also, flow cytometry will be performed to follow changes in cell
viability on a daily
basis for the 1-week period. For the sampling purpose and medium
replenishment, a sampling
port will be added in the external circulation loop.
[00346] After the empty device characterization, how the presence of CRIECs
and
APCs (i.e., DCs) in these chambers will interfere with the movement of T cells
will be
studied. For the epithelial culture chamber, experiments after CRIECs reach
confluence will
be performed, which initial observations indicate takes about 1-4 days.
[00347] In preliminary experiments, no evidence of m-CRIECs blocking T cell
movements was seen, although such observations to date are limited. The flow
rate and
biomaterial parameters will be optimized to ensure that >80% can freely be
recirculated
through the chambers for up to 1 week.
[00348] Experiments to establish that T cells become activated and persist for
3
weeks due to biomimetic recirculation. Due to the recirculatory attribute of
iGVHD, G1
miHA-specific T cells continuously stimulated in the APC and tissue chambers
are expected
to persist and expand over the 3-week benchmark period to cause measurable
CRIEC
damage. The operation of the APC chamber that can be initially seeded with 105
DCs
(sufficient for the stimulation of 106 T cells) will be optimized. m-DCs will
be prepared by
injecting host mice with B16-FLt3L tumor, which promotes the enrichment of DCs
in tumor
bearing mice. (Anandasabapathy, N. et al. Classical F1t3L-dependent dendritic
cells control
immunity to protein vaccine. J. Exptl Med. 211, 1875-1891,
doi:10.1084/jem.20131397
(2014); Anandasabapathy, N. et al. F1t3L controls the development of
radiosensitive dendritic
cells in the meninges and choroid plexus of the steady-state mouse brain. J.
Exptl Med. 208,
1695-1705, doi: 10.1084/jem.20102657 (2011)).
[00349] Human DCs (h-DCs) will be derived from patient PBL monocytes
(Santodonato, L. et al. Monocyte-derived dendritic cells generated after a
short-term culture
with IFN- alpha and granulocyte-macrophage colony-stimulating factor stimulate
a potent
Epstein-Barr virus- specific CD8+ T cell response. J. Immunol. 170, 5195-5202
(2003)).
[00350] The following experiments will be performed with murine ceils first
and
later confirmed with human cells. As preliminary results suggest, m-DCs can
infiltrate into
the microbeads assembly and become adhered to the microbead surface. Upon the
introduction of T cells and their physical contact, T cells will be activated.
Since DCs are
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programmed to die after maturation (typically within 5 days) and therefore in
order to provide
constant stimulations (Abdi, K., Singh, N. J. & Matzinger, P.
Lipopolysaccharide-activated
dendritic cells: "exhausted" or alert and waiting? J. Immunol. 188, 5981-5989,
doi:
10.4049/jimmuno1.1 102868 (2012)) the capability to replenish dead DCs with
new DCs will
need to be developed. Simply adding new DCs at 5-day intervals by opening the
PDMS
plug and placing them onto the top of the microbeads assembly is planned. It
is expected that
dead cell debris will be washed away and the microbead surface will become
available again
for the arrival and adhesion of new DCs, since T cells do not adhere to the
microbead surface
(as observed in preliminary experiments). The effectiveness of the
replenishment approach at
providing constant T cell stimulation will be evaluated by measuring cell
viability, activation,
and proliferation at various replenish time intervals (3, 7, 14, 21 days) over
3 weeks. After the
APC chamber is optimized, the synergistic effects of CRIECs on T cell
viability, activation,
and proliferation will be investigated and compared with DCs only. Annexin
V/PI staining
will be used to determine the viability of T cells. T cell activation will be
determined by
percent changes in CD25 and CD69 expressions. For the proliferation assay
(Zhang, W., Lee,
W. Y., Siegel, D. S., Tolias, P. & Zilberberg, J. Patient-Specific 3D
Microfluidic Tissue
Model for Multiple Myeloma. Tissue Engineering. Part C, Methods 20, 663-670,
doi:
10.1089/ten.TEC.2013.0490 (2014)), T cells will be labeled with cell trace
carboxyfluorescein succinimidyl ester (CFSE) proliferation dye and analyzed
using flow
cytometry.
[00351] If DCs cannot be replenished by the infiltration approach, replacing
the
whole assembly and place a new microbead/dendtric cell assembly with T cells
separated
from the old assembly and re-introduced will be considered. Also, cytokines
(e.g., IL-2) can
be added to the culture chamber in order to prolonged T cell maintenance
(Hedfors, I. A. &
Brinchmann, J. E. Long-term proliferation and survival of in vitro-activated T
cells is
dependent on Interleukin-2 receptor signalling but not on the high-affinity IL-
2R.
Scandinavian journal of immunology 58, 522-532 (2003)). Moreover, retinoic
acid could be
added to generate gut-tropic DCs, which should facilitate the generation of T
cells with
superior 'EC-killing avidity (Gorfu, G., Rivera-Nieves, J. & Ley, K. Role of
beta7 integrins
in intestinal lymphocyte homing and retention. Current Molec. Med. 9, 836-850
(2009);
Agace, W. W. T-cell recruitment to the intestinal mucosa. Trends in Immunol.
29, 514-522,
doi: 10.1016/j.it.2008.08.003 (2008)).
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[00352] The relatively large hole-to-hole distance in the current membrane
material
may limit the T cell movement through the membranes. Although this was not
seen in
preliminary experiments with 106 circulating T cells, this may be an issue
when the number
of circulating T cells is significantly increased to achieve high E:T ratios.
If this becomes a
problem, using polyethylene terephthalate (PET) membrane (Greiner Bio-One)
with the
average pore size of 8 p.m and the surface pore density of 1.5x106 cm-2 (vs.
105 cm-2 for the
current PC membrane) will be considered.
[00353] Experiments to Establish that iGVHD can facilitate CRIECs killing
within 2 or 3 weeks: After operative procedures are optimized from the above
tasks, iGVHD
will be used to determine PIdx values using cells from murine GVHD models
(Table 1). T
cell recirculation is expected to: (1) lower the E:T cell ratio (i.e., the
seeding ratio of CRIECs
and T cells in iGVHD) to achieve measurable m-CR1EC killing and (2) speed up
the killing
for the reasons articulated earlier. For these experiments, the E:T ratios
will be titrated in the
range of 2:1 to 20:1. Because of the plate reader assay capability of the
platform,
measurement of % cell death for calculation of PIdx is expected to be
streamlined using in
situ determination of cell death by caspase-3 staining (Luft, T. et al. Serum
cytokeratin-18
fragments as quantitative markers of epithelial apoptosis in liver and
intestinal graft-versus-
host disease. Blood 110,4535-4542, doi: 10.1182/blood-2006-10-049817 (2007);
Disbrow, G.
L. et al. Dihydroartemisinin is cytotoxic to papillomavirus-expressing
epithelial cells in vitro
and in vivo. Cancer Res. 65, 10854-10861, doi:10.1158/0008-5472.CAN-05-1216
(2005)).
After iGVHD's facilitated killing capability is established with murine cells,
the results will
be confirmed using patient- derived cells. Based on these results, overall
iGVHD design
features and operational protocols will be reviewed and revised as necessary.
[00354] Experiments to Correlate statistically GVHD risk predictions from
iGVHD from 24 patient-donor samples with patient outcomes: For each patient-
donor pair,
PIdx will be determined using the iGVHD device and the protocols developed in
the previous
tasks. The recirculation and high-throughput capabilities of the device will
be utilized to
evaluate 3 or more E:T ratios. PIdx values determined from 24 patient-donor
samples will be
compared to patient outcomes as follows. The main outcome of interest,
severity of GVHD
(grades 0-IV), will be dichotomized in low severity (LS:0-1) and high severity
(HS: 11, III,
IV). Discriminant validity of PIdx will be examined by comparing PIdx from LS
and HS
groups using a two-sided Student's t-test or Wilcoxon rank sum test, as
appropriate. Logistic
regression analysis will be performed to assess the capability of PIdx as a
risk predictor of
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GVHD. The results of this analysis will be presented as odds ratios (OR), 95%
confidence
interval, P-value. Area under receiver operating characteristics (ROC) curve
will be used to
quantify probability of accurate classification of LS vs. HS outcomes. ROC
analysis and
optimal cut point function based on Youden Index will be used to determine the
cutoff value
for PIdx. Hochberg procedure will be utilized to adjust for multiple testing.
Using the
determined cutoff value, sensitivity, specificity, positive predictive value
(PPV) and negative
predictive value (NPV), overall accuracy will be calculated and reported using
standard 2 by
2 tables for categorical analysis.
[00355] Follow-On Clinical Study. A follow-on clinical study with prospective
sample collection is anticipated after proof of principle of predicting GVHD
using human
samples is established. Samples of 3 to 4 donors for haploidentical cases can
be screened,
although corroboration of the predictive outcome will be only for the selected
haploidentical
donor. Nonetheless, these cases will be use as a proof-of-principle that
responses between
multiple donors using this approach can be discerned.
Example 9
Circulation of Primary Murine T Cells Through Primary Murine Intestine
Epithelial Cells
Maintained on Nanofibrous Mesh:
[00356] Adoptive T cell therapy in the form of allogeneic blood and marrow
transplantation (allo-BMT) has proven to be one of the few curative treatments
for patients
suffering from a number of drug-resistant hematological malignancies. However,
the full
exploitation of this clinical intervention is greatly limited by graft versus
host disease
(GVHD), as one of the major BMT complications. This disease is characterized
by severe
and potentially lethal tissue damage to skin, liver, and gut tissues of
transplanted patients,
mediated by donor T cells responding to host alloantigens.35-37 In particular,
GVHD of the
gastrointestinal tissues is closely associated with non-relapse mortality
following allo-BMT
(A. C. Harris, J. E. Levine and J. L. Ferrara, Clin. Haematol., 2012, 25, 473-
478). Currently,
there is no way to predict which patient¨donor pairs will develop GVHD after
BMT. Our
long-term interest is to explore the possibility of emulating the potential
killing of patient-
derived intestinal epithelial cells (IECs) by donor T cells, where IECs are
the primary
population targeted in GI GVHD (A. M. Hanash, J. A. Dudakov, G. Hua, M. H.
O'Connor, L.
F. Young, N. V. Singer, M. L. West, R. R. Jenq, A. M. Holland, L. W. Kappel,
A. Ghosh, J.
J. Tsai, U. K. Rao, N. L. Yim, O. M. Smith, E. Velardi, E. B. Hawryluk, G. F.
Murphy, C.
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Liu, L. A. Fouser, R. Kolesnick, B. R. Blazar and M. R. M. van den Brink,
Immunity, 2013,
37, 339-350; R. El-Asady, R. Yuan, K. Liu, D. Wang, R. E. Gress, P. J. Lucas,
C. B.
Drachenberg and G. A. Hadley, J. Exp. Med., 2005, 201, 1647-1657). In native
tissues, IECs
reside on a thin fibrous basement membrane (BMa) consisting of the
intermingled networks
of laminins and collagens and provides cell anchoring and barrier functions.
The membrane
networks interact with cells through membranous integrin receptors and other
plasma
membrane molecules, influencing cell differentiation, migration, adhesion,
phenotype, and
survival.
[00357] As an initial step towards this application, we used our prototype
device
to: (1) culture and maintain primary conditionally reprogrammed murine IECs
isolated from
the small intestine of a C57B1/6-TgIJCAG-OVA)916 Jen/J mouse (B6-SIINFEKL) and
(2)
assess the device's capability in supporting the circulation of primary murine
T cells through
the IECs (FIG. 9a for conceptual illustration of the experimental approach).
[00358] As shown by the scanning electron microscopic (SEM) image in FIG. 9b,
nanofiber mesh was used to mimic the BMa of the epithelial tissue as well as
to support the
long-term viability of IECs. The latter role is particularly important, since
primary IECs
cannot be kept viable during conventional culture. The nanofiber mesh was
produced by
electronspinning polycaprolactone (PCL)/type I collagen onto the PC membrane
surface prior
to the device assembly (X. Yang, K. R. Ogbolu and H. Wang, J. Exp. Nanosci.,
2008, 3, 329-
345). The average open space in the nanofiber mesh was controlled to about
61.tm (FIG. 9b)
since the average diameter of T cells is about 5 pm. For the same reason, we
also selected the
average pore of the PC membrane to be 101.tm (FIG. 9b). With the use of
nanofiber mesh,
IECs were able to develop into a confluent layer and exhibit cobblestone
morphology while
remaining viable in the perfusion device for up to 7 days (FIG. 9c).
[00359] After IECs became confluent (approximately 4 days post seeding),
enriched T cells obtained from an eGFP transgenic C57B16/J mouse were
introduced and
circulated through the culture chambers (2.5 x 105 cells per chamber). As
illustrated in FIG.
9a, a peristaltic pump was used to circulate T cells in RPMI complete medium.
Visually, we
did not see the entrapment of T cells in any part of the culture device and
external circulatory
pathways. T cell viability was quantified by sampling the culture medium at
various time
points and counting live and dead T cell numbers suspended in the medium. As
shown in
FIG. 9d, the overall viability of T cells decreased during the 72 h culture
period. This was
expected since it is well known that the viability of T cells cannot be
maintained in vitro
79
CA 02972846 2017-06-29
WO 2016/112245 PCT/US2016/012573
unless they are stimulated by antigen-presenting cells or maintained via the
addition of
cytokines like IL-2 (P. Marrack and J. Kappler, Annu. Rev. Immunol., 2004, 22,
765-787).
However, interestingly, there were more viable T cells when they were
circulated through the
IEC layer. The results suggest that T cells were activated by IECs, resulting
in the increased
viability of T cells. Although both B6-SIINFEKL IECs and T cells were of B6
background,
it is likely that minor antigen differences between the B6-SIINFEKL and the
eGFP-B6 strains
could have elicited activation of T cells and potentially other cells like
natural killer (NK)
cells. FIG. 9e shows that IECs were spreading on the nanofiber mesh surface,
and were in
physical contact with T cells. Since the membrane pores (10 Ilm) and opening
spaces (>6 Ilm)
between nanofibers were larger than the T cells (5 lm), they were able to go
through the IEC
layer without getting trapped in the culture chamber.
[00360] While the described invention has been described with reference to the
specific embodiments thereof it should be understood by those skilled in the
art that various
changes may be made and equivalents may be substituted without departing from
the true
spirit and scope of the invention. In addition, many modifications may be made
to adopt a
particular situation, material, composition of matter, process, process step
or steps, to the
objective spirit and scope of the described invention. All such modifications
are intended to
be within the scope of the claims appended hereto.
