Note: Descriptions are shown in the official language in which they were submitted.
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INHIBITION OF GRAFT versus HOST DISEASE
FIELD OF THE INVENTION
This invention relates to cellular compositions useful in medical
treatments, processes for their preparation and their uses in medical
treatments.
More specifically, it relates to cellular compositions useful in alleviation
of
complications following allogeneic bone marrow transplantation, namely graft
versus host disease in mammalian patients, especially in human patients, and
to
processes for preparation of such compositions of matter.
BACKGROUND OF THE INVENTION
Bone marrow transplantation, BMT, is indicated following a process
which destroys bone marrow. For example, following intensive systemic
radiation
or chemotherapy, bone marrow is the first target to fail. Metastatic cancers
are
commonly treated with very intensive chemotherapy, which is intended to
destroy
the cancer, but also effectively destroys the bone marrow. This induces a need
for
BMT. Leukemia is a bone marrow malignancy, which is often treated with BMT
after chemotherapy and/or radiation has been utilized to eradicate malignant
cells.
BMT is currently used for treatment of leukemias which are life-threatening.
Some
autoimmune diseases may be severe enough to require obliteration of their
native
immune systems which includes concomitant bone marrow obliteration and
requires subsequent bone marrow transplantation. Alleviation of any but the
most
acute life-threatening conditions involving bone marrow disorders with BMT is,
however, generally regarded as too risky, because of the likelihood of the
onset of
graft versus host disease.
Graft-versus-host disease, GVHD, is an immunological disorderthat
is the major factor that limits the success and availability of allogeneic
bone
marrow or stem cell transplantation (collective referred to herein as alto-
BMT) for
treating some forms of otherwise incurable hematological malignancies, such as
leukemia. GVHD is a systemic inflammatory reaction which causes chronic
illness
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and may lead to death of the host mammal. At present, allogeneic transplants
invariably run a severe risk of associated GVHD, even where the donor has a
high
degree of histocompatibility with the host.
GVHD is caused by donor T-cells reacting against systemically
distributed incompatible host antigens, causing powerful inflammation. In
GVHD,
mature donor T-cells that recognize differences between donor and host become
systemically activated. Current methods to prevent and treat GVHD involve
administration of drugs such as cyclosporin-A and corticosteroids. These have
serious side effects, must be given for prolonged periods of time, and are
expensive to administer and to monitor. Attempts have also been made to use T-
cell depletion to prevent GVHD, but this requires sophisticated and expensive
facilities and expertise. Too great a degree of T-cell depletion leads to
serious
problems of failure of engraftment of bone marrow stem cells, failure of
hematopoietic reconstitution, infections, or relapse. More limited T-cell
depletion
leaves behind cells that are still competent to initiate GVHD. As a result,
current
methods of treating GVHD are only successful in limited donor and host
combinations, so that many patients cannot be offered potentially life-saving
treatment.
BRIEF REFERENCE TO THE PRIOR ART
International Patent Application No. PCT/CA97/00564 Bolton
describes an autovaccine for alleviating the symptoms of an autoimmune disease
in a mammalian patient, comprising an aliquot of modified blood obtained from
the
same patient and treated extracorporeally with ultraviolet radiation and an
oxygen/ozone gas mixture bubbled therethrough, at an elevated temperature
(42.5°C), the autovaccine being re-administered to the same patient
after having
been so treated.
It is an object of the present invention to provide a process of
alleviating the development of GVHD complications in a mammalian patient
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undergoing allo-BMT procedures.
SUMMARY OF THE INVENTION
According to the present invention, a patient being treated by allo-
BMT is administered a composition containing T-cells obtained from an
allogeneic
donor, said T-cells having been subjected in vitro to oxidative stress to
induce
therein decreased inflammatory cytokine production coupled with reduced
proliferative response. It appears that such oxidatively stressed allogeneic T-
cells
when injected into a mammalian patient, have a down-regulated immune response
and a down-regulated destructive allogeneic response against the recipient, so
that engraftment of the hematopoietic stem cells, administered along with or
separately from the stressed T-cells, can take effect with significantly
reduced risk
of development of GVHD. The population of stressed T-cells nevertheless
appears
to be able to exert a sufficient protective effect on the mammalian system to
guard
against failure of engraftment and against infection, whilst the hematopoietic
system is undergoing reconstitution, at least in part, by proliferation and
differentiation of the allogeneic stem cells.
One aspect of the present invention provides, accordingly, a process of
treating a
mammalian- patient for alleviation of a bone marrow mediated disease, with
alleviation of consequently developed graft versus host disease (GVHD), which
comprises administering to the patient allogeneic hematopoietic stem cells and
allogeneic T-cells, at least a portion of said T-cells having been subjected
to
oxidative stress in vitro, prior to administration to the patient, so as to
induce an
altered cytokine production profile and a reduced proliferative response
therein.
Another aspect of the present invention provides a population of mammalian T-
cells, essentially free of stem cells, said T-cells having been subjected in
vitro to
oxidative stress so as to induce in said cells an altered cytokine production
profile
and a reduced proliferative response.
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A further aspect of the present invention provides a process for preparing an
allogeneic cell population for administration to a human patient suffering
from a
bone marrow mediated disease, which comprises subjecting, in vitro, a
population
of donor cells enriched in T-cells to oxidative stress to induce in said T-
cells an
altered cytokine production profile and a reduced proliferative response.
BRIEF REFERENCE TO THE DRAWINGS
FIGURES 1 and 2 of the accompanying drawings are graphical
presentations of results obtained according to Example 3 below.
FIGURE 3 is a depiction of the results obtained from Example 4
below.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The process of the present invention involves an initial collection of
hematopoietic stem cells and T-cells from a donor. The preferred source of
such
cells is mobilized stem cells and T-cells from the peripheral blood of the
donor.
Stem cells are present in very small quantities in peripheral blood, and one
preferred way of operating in accordance with the invention is to enrich the
stem
cell population of the donor's peripheral blood, and then to extract the
donor's
peripheral blood for use as a source of stem cells and T-cells for treatment
as
described and subsequent injection into the patient. Enrichment may be
achieved
by giving the donor a course of injections of appropriate growth factors, over
several days e.g. fve days prior to extracting peripheral blood from the
donor.
Appropriate cell fractions can be collected from the blood by leukopheresis, a
known procedure, as it is extracted, with the plasma and red cells being
returned
to the donor, in a closed flow system. The white cell collection, which
contains the
stem cells (about 3%) and T-cells (about 40%) along with B-cells, neutrophils
and
other white cells, may be treated to alter their cytokine production profiles
and to
reduce the proliferative response of the T-cells therein, and then
administered to
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the host patient, in accordance with the invention, as a whole collection of
cells
(peripheral blood mononuclear cells). Preferably, however, the donor T-cells
are
separated from the other cells; so that only the T-cells are subjected to
oxidative
stress and then administered to the patient, with the stem cells for
engraftment
being administered to the patient separately from the treated T-cells. For
practical
purposes, however, subjection of the collection of peripheral blood
mononuclear
cells to the stressors is satisfactory, without further fractionation to
isolate the T-
cells, which is a difficult and expensive procedure. Separate administration
of stem
cells is strongly preferred.
If for some reason it is desired to subject the entire white cell
collection to oxidative stress to induce the aforementioned changes in the T-
cell
portion thereof, and then administer the entire collection to the patient, it
is
preferred to protect the stem cells from any damaging effects of the oxidative
stress in a manner described below.
In an alternative, but less preferred, procedure, whole bone marrow
of the donor can be used as the source of T-cells and stem cells for the
process
of the invention. Whole bone marrow has in the past been the usual source of
cells for allogeneic cell transplantation procedures, and can indeed be used
in the
present process. It is however an inconvenient and uncomfortable procedure for
the donor, requiring anaesthetic and lengthy extraction procedures. Any source
of
T-cells and stem cells from the donor.can be used in principle, but peripheral
blood
enriched in stem cells and T-cells is the most clinically convenient.
The alteration in cytokine production profile induced in the T-cells in
the process of the invention is preferably a reduction in production of
inflammatory
cytokines, such as interferon-y and tissue necrosis factor-a.
The oxidative stress may be applied to the T-cells by subjecting
them to an oxidative environment such as the addition of a gaseous, liquid or
solid
chemical oxidizing agent (ozone, molecular oxygen, ozoneloxygen gas mixtures,
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permanganates, periodates, peroxides, drugs acting on biological systems
through
an oxidative mechanism such as adriamycin, and the like). In one preferred
method according to the invention, the T-cells are subjected, in suspension,
to a
gaseous oxidizing agent, such as an ozone/oxygen gas mixture bubbled through
the suspension of cells, optionally in combination with the simultaneous
subjection
of the cells to ultraviolet radiation, in appropriate doses.
One method according to the present invention subjects the
allogeneic white cells from the donor, including both the stem cells and the T-
cells,
to oxidative stress. This eliminates the need to include a complicated and
costly
step of separating the T-cells from the other cellular components of the white
cells
composition. In such case, however, it is strongly preferred to protect the
stem
cells in the composition from deleterious effects of the stress. This can be
accomplished by including one or more stem cell growth factors in the cell
composition at the time of subjecting it to the stress. Protection of the stem
cells
from the deleterious effects of the oxidative stress is achieved by the
presence of
growth factors, and so, prior to subjecting the stem cell-T-cell composition
to
oxidative stress, one or more stem cell growth factors are added to the
composition. Stem cell growth factors useful in the process are cytokines
which
promote survival of stem cells (but not T-cells) during this stressing. They
are
cytokines which interact with growth receptors on stem cells. They are
believed
to activate the MAP-kinase pathway of the cell, resulting in the activation of
erk.
Examples of suitable such growth factors, include stem cell specific growth
factors,
kit-ligand, IL-3, GM-CSF and FLT 3 ligand, all of which are known. It is
preferred
to add precise amounts of extracted, purified growth factors or, especially,
recombinant growth factors available on the market, or combinations thereof,
suitably dissolved or suspended in appropriate, biologically acceptable
fluids.
One preferred method of subjecting the allogeneic T-cells to
oxidative stress according to the invention involves exposing a suspension of
the
cells to a mixture of medical grade oxygen and ozone gas, for example by
bubbling
through the suspension a stream of medical grade oxygen gas having ozone as
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a minor component therein. The suspending medium may be any ofthe commonly
used biologically acceptable media which maintains cells in viable condition.
The
ozone gas may be provided by any conventional source known in the art.
Suitably
the gas stream has an ozone content of from about 1.0-100 ,ug/ml, preferably 3-
70
~cglml and most preferably from about 5-50 ~cg/ml. The gas stream is supplied
to
the aliquot at a rate of from about 0.01-2 litres per minute, preferably 0.05-
1.0 litres
per minute, and most preferably at about 0.06-0.30 litres per minute (STP).
Another method of subjecting the T-ce(Is to oxidative stress to render
them suitable for use in the present invention is to add to a suspension of
the cells
a chemical oxidant of appropriate biological acceptability, and in
biologically
acceptable amounts. Permanganates, periodates and peroxides are suitable,
when used in appropriate quantities. Hydrogen peroxide is useful in
demonstrating
the effectiveness of the process of the invention and in giving guidance on
the
appropriate quantity of oxidizing agent to be used, although it is not an
agent of
first choice forthe present invention, for practical reasons. Thus, a suitable
amount
of oxidizing agent is hydrogen peroxide in a concentration of from 1
micromolar -
2 millimolar, contacting a 10 ml suspension containing from 10'~ to 10$ cells
per
ml, for 20 minutes, or equivalent oxidative stress derived from a different
oxidizing
agent. Optimum is about 1 millimolar hydrogen peroxide in such a suspension
for
about 20 minutes, or the equivalent of another oxidizing agent calculated to
give
a corresponding degree of oxidative stress to the cells.
The size of the cell suspension to be subjected to oxidative stress
is generally from about 0.1 ml to about 1000 ml, preferably from about 1-500,
and
containing appropriate numbers of T-cells for subsequent administration to a
patient undergoing also-BMT. These numbers generally correspond to those used
in prior methods of allogeneic T-cell administration in connection with alto-
BMT,
and are familiar to those skilled in the art.
One specific process according to the invention is to subject the cell
suspension simultaneously to oxygen/ozone bubbled through the suspension and
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ultraviolet radiation. This also effects the appropriate changes in the nature
of the
T-cells. Care must be taken not to utilize an excessive dosage of oxygen/ozone
or
UV, to the extent that the cell-membranes are caused to be disrupted, or other
irreversible damage is caused to an excessive number of the cells.
The temperature at which the T-cell suspension is subjected to the
oxidative stress does not appear to be critical, provided that it keeps the
suspension in the liquid phase and is not so high that it causes cell membrane
disruption. The temperature should not be higher than about 45°C.
When ultraviolet radiation is used in conjunction with the
oxygen/ozone oxidative stressor, it is suitably applied by irradiating the
suspension
under treatment from an appropriate source of UV radiation, while the aliquot
is
maintained at the aforementioned temperature and while the oxygen/ozone
gaseous mixture is being bubbled through the aliquot. The ultraviolet
radiation
may be provided by any conventional source known in the art, for example by a
plurality of low-pressure ultraviolet lamps. There is preferably used a
standard UV-
C source of ultcaviolet radiation, namely UV lamps emitting primarily in the C-
band
wavelengths, i.e. at wavelengths shorter than about 280 nm. Ultraviolet
radiation
corresponding to standard UV-A and UV-B sources can also be used. Preferably
employed are low-pressure ultraviolet lamps that generate a line spectrum
wherein
at least 90% of the radiation has a wavelength of about 254 nm. An appropriate
dosage of such UV radiation, applied simultaneously with the aforementioned
temperature and oxidative environment stressors, is obtained from lamps with a
power output of from about 5 to about 25 watts, preferably about 5 to about 10
watts, at the chosen UV wavelength, arranged to surround the sample container
holding the aliquot. Each such lamp provides an intensity, at a distance of 1
metre, of from about 40 - 80 micro watts per square centimeter. Several such
samples surrounding the sample container, with a combined output at about 254
nm of 15 - 40 watts, preferably 20 - 40 watts, operated at maximum intensity
may
advantageously be used. At the incident surface of the aliquot, the UV energy
supplied may be from about 0.25 -4.5 j/cmz during a 3-minute exposure,
preferably
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0.9 - 1.8 jlcm2. Such a treatment provides a suspension aliquot which is
appropriately modified according to the invention ready for injection into the
patient. -
The time for which the aliquot is subjected to the stressors can be
from a few seconds to about 60 minutes. It is normally within the time range
of
from about 0.5 - 60 minutes. This depends to some extent upon the chosen
intensity of the UV irradiation, the temperature and the concentration of and
rate
at which the oxidizing agent is supplied to the aliquot. Some experimentation
to
establish optimum times and dosages may be necessary on the part of the
operator, once the other stressor levels have been set. Under most stressor
conditions, preferred times will be in the approximate range of about 0.5 - 10
minutes, most preferably 2 - 5 minutes, and normally around 3 minutes.
In the practice of one preferred process of the present invention, the
suspension of cells may be treated with oxygenlozone gas mixture and
optionally
also with UV radiation using an apparatus of the type described in U.S. patent
4,968,483 Mueller. The suspension is placed in a suitable, sterile, UV-
radiation-
transmissive container, which is then fitted into the machine. The temperature
of
the aliquot is adjusted to the predetermined value, e.g. 42.511 °C, by
the use of a
suitable heat source such as an IR lamp, and the UV tamps are switched on for
a
fixed period before the gas flow is applied to the aliquot providing the
oxidative
stress, to allow the output of the UV lamps to stabilize. The oxygen/ozone gas
mixture, of known composition and control flow rate, is applied to the
aliquot, for
the predetermined duration of 0.5 - 60 minutes, preferably 1 - 5 minutes and
most
preferably about 3 minutes as discussed above. In this way, the suspension is
appropriately modified according to the present invention sufficient to
achieve the
desired effects of alleviation or prevention of GVHD.
From another aspect, the preferred embodiment of the present
invention may be viewed as a process of treating allogeneic T-cells prior to
their
introduction into a patient, by extracorporeally stressing the T-cells, which
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comprises subjecting the T-cells to oxidative stress such as exposure to ozone
or
ozoneloxygen. The treated allogeneic T-cells from the process of the invention
hive a direct effect on the development and progression of GVHD. The donor T-
cells pretreated according to the process of the invention prior to
introduction into
the host patient, have been modified, so that they no longer mount a
deleterious
response. Their ability to mount an inflammatory cytokine response has been
decreased. For example their ability to secrete IFNy, TNFa and IL-2, and their
proiiferative response to standard mitogens has been reduced. Accordingly they
no longer react against incompatible systemically distributed host
histocompatibility
antigens to cause inflammation to any great extent. The allogeneic stem cells
administered to the patient can proceed with engraftment with improved chance
of success. After a period of time, the treated T-cells largely recover their
proliferative ability and immune response functions, but remain relatively
unresponsive (tolerant) to differing host histocompatibility antigens.
The invention is further described, for illustrative purposes, in the
following specific examples.
SPECIFIC DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS
The spleen of a mamma! offers a convenient, accessible source of
cells, especially T-cells but also including small quantities of stem cells
and is
particularly useful in connection with animal models for experimental
purposes.
Experimental testing to obtain indication of the utility of the process
of the present invention was conducted using a model of acute GVHD in SCID
mice. T-cells from C57B1/6J (B6) mice were intravenously injected into sub-
lethally irradiated CB-17 SCID mice. The latter are congenitally lymphopenic
and
provide a strong stimulus for donor cells due to their complete disparity at
the
major histocompatibility locus (MHC). The mean survival time of host mice in
this
model is 14 days. GVHD is characterized by suppression of host hematopoietic
recovery from irradiation; expansion of T-cells that use V~3 chain to form
their T-
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cell receptor complexes (TCR's); spontaneous secretion of interferon-y and TNF
-a, by donor T-cells, and aberrant localization of donor T-cells to the red
pulp
areas of the spleen. If donor marrow is co-injected with T-cells, a chronic
form of
GVHD results.
EXAMPLE 1
Mouse spleen cells from C57B1l6J (B6) mice were suspended to a
density of 10'Iml in a-MEM, 2ME and 10% fetal calf serum (FCS). The FCS
contains cytokines and growth factors. The cell suspension was subjected
simultaneously to ultraviolet radiation from UV-C lamps, wavelength 253.7 nm,
whilst bubbling through the suspension a gas mixture of 14 - 15 mcglml
ozone/medical grade oxygen, at 42.5°C. The treatment took place for 3
minutes.
Immediately after the treatment, the cells had a viability of only about
10%.
EXAMPLE 2
The experiment of Example 1 was essentially repeated except that
the cells were suspended in 100% FCS. The immediate survival of the cells in
this
case was 50 - 60%, indicating that factors present in the FCS have exerted a
protective effect on at least some of the cells.
EXAMPLE 3
Murine B6 spleen cells suspended in 100% FCS were subjected to
UV-oxidation-heat treatment The cell suspension was subjected simultaneously
to ultraviolet radiation from UV-C lamps, wavelength 253.7 nm, whilst bubbling
through the suspension a gas mixture of 14 - 15 mcg/ml ozone/medical grade
oxygen, at 42.5°C. The treatment took place for 3 minutes. Varying
numbers were
injected into sub-lethally irradiated CB-17 SCID mice. Their subsequent
behaviour
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was compared with similar numbers of B6 spleen cells, not subjected to the
treatment.
Figure 1 is a graphical presentation of the results of these
experiments, where the % survival of the animals in each group is plotted as
ordinate against days following injection of the treated or untreated cells.
At all
dosage levels, there is a marked improvement of survival when the treated
cells
are used as opposed to the untreated cells, demonstrating potential for the
process of the invention in alleviating GVHD.
Figure 2 of the accompanying drawings is a plot of the number of
donor T-cells per spleen against days after GVHD induction, in these same
experiments. This shows that the treated donor T-cells survive and expand in
number in the host mice, although to a more limited degree than control,
untreated
B6 T-cells.
EXAMPLE 4
Six days after initiation of GVHD in the mice by injection of the donor
cells (treated and untreated), donorT-cells were separated from SCID spleen
cells
by density gradient centrifugation. Intracellular cytokine staining was
performed
according to the method of Ferrick, D.A. et. al., NATURE 373 225,257, 1995.
The
staining was performed on spleen cell suspensions on day 8 after injection of
B6
spleen cells. Cytokine production was determined 4 hours after stimulation in
vitro
with PHA and ionomycin in the presence of brefeldin-A and after gating on CD4+
and CD8+. The results were assessed by intracellular flow cytometry, and the
results thereof are depicted in Fig. 3 of the accompanying drawings. The
percentage of each cells in each quadrant is recorded. The drawing shows
significantly reduced levels of the inflammatory cytokines interferon-y (INF)
and
tissue necrosis factor-a (TNF), lower right quadrants, from the T-cells which
had
been stressed as described in Example 1, as compared with untreated cells and
controls.
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EXAMPLE 5
Inversion of the normal ratio of CD4+ to CD8+ T-cells (usually
approximately 2:1 ) is known to accompany GVHD. By intracellular cytokine
staining techniques following the method of Ferrick et.al., Nature 373: 255-
257,
1995 and using anti-CD4 and CD8-tricolor antibodies, CD41CD8 ratios were
determined. In the untreated donor spleen cells after injection into sub-
lethally
irradiated mice, the inversion of the normal ratio was confirmed. The initial
CD4lCD8 ratios of 1.310.2 and 2.210.3 decreased to 0.3310.05 and 0.9~0.1 by
day13 for unstressed B6 and C3H donor T cells, respectively, at a time when
many
animals were succumbing to GVHD. In contrast, the ratios remained greater than
1 at all times and correlated with the lack of clinical evidence of GVHD when
donor cells had been pretreated with the stressors as described in Example 1.
EXAMPLE fi
This example demonstrates the principle of the invention, using
oxidative stress alone, provided by hydrogen peroxide, an effective chemical
oxidizing agent and representative of many other, perhaps more biologically
suitable, chemical oxidizing agents.
Peripheral human blood mononuclear cells PBMCs, which is a
collection of white blood cells comprising about 40% T-cells, were stressed by
contact with aqueous solutions of hydrogen peroxide, of various
concentrations,
for 20 minutes. Their immediate survival was measured, along with their
immediate
phytohaemagglutinin (PHA) response. Then their survival after 24 hours was
measured, followed by their PHA response (tritiated thymidine uptake following
mitogenic stimulation with PHA) and cytokine profile after 7 days. The results
are
given in the following table.
TABLE
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Conc. H202 Immediate 24 hr survival immediatePHA response Cytokine
survival % % PHA respon se 7-day Profile
100pmole/L 80-90 100 2000 + IFNyI
300Nmole/L 80-90 50 2000 + IFNyI
1 mmole/L 80-90 40 400 + IFNY1
3 mmole/L 80-90 40 400 + IFNy1
Control 95 95 8575 + IFNyt
These results indicate that
T-cells subjected to oxidative
stress alone
achieve creased proliferative response
a de and decreased inflammatory
cytokine
production,
suitable for
use in the
present invention.
