U.S. patent application number 15/267417 was filed with the patent office on 2017-01-05 for human facilitating cells.
The applicant listed for this patent is University of Louisville Research Foundation, Inc.. Invention is credited to Mary Jane Elliott, Suzanne T. Ildstad.
Application Number | 20170000825 15/267417 |
Document ID | / |
Family ID | 45605659 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170000825 |
Kind Code |
A1 |
Ildstad; Suzanne T. ; et
al. |
January 5, 2017 |
HUMAN FACILITATING CELLS
Abstract
The present disclosure relates to human facilitating cells
(hFC), and methods of isolating, characterizing, and using such
hFCs.
Inventors: |
Ildstad; Suzanne T.;
(Prospect, KY) ; Elliott; Mary Jane; (Brandenburg,
KY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Louisville Research Foundation, Inc. |
Louisville |
KY |
US |
|
|
Family ID: |
45605659 |
Appl. No.: |
15/267417 |
Filed: |
September 16, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14157888 |
Jan 17, 2014 |
9452184 |
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15267417 |
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12957011 |
Nov 30, 2010 |
8632768 |
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14157888 |
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PCT/US2009/003340 |
Jun 1, 2009 |
|
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12957011 |
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61057724 |
May 30, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 7/06 20180101; A61P 25/28 20180101; A61P 7/00 20180101; A61P
37/00 20180101; A61P 31/14 20180101; C12N 5/0634 20130101; C12N
5/0647 20130101; A61P 35/02 20180101; A61P 25/00 20180101; A61K
35/17 20130101; A61K 35/28 20130101; A61K 35/12 20130101; C12N
5/0652 20130101; A61P 37/02 20180101; A61P 3/10 20180101; A61P 1/16
20180101; A61P 31/18 20180101; A61P 31/12 20180101; A61P 3/00
20180101; A61P 35/00 20180101 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12N 5/0789 20060101 C12N005/0789 |
Claims
1. A cellular composition comprising at least about 30% human
facilitating cells (hFCs), wherein said hFCs comprise cells having
a phenotype of CD8+/alpha beta TCR-/CD56.sup.dim/neg and cells
having a phenotype of CD8+/alpha beta TCR-/CD56.sup.bright.
2. The cellular composition of claim 1, wherein said cells having a
phenotype of CD8+/alpha beta TCR-/CD56.sup.dim/neg are
predominantly CD3 epsilon+/CD19-.
3. The cellular composition of claim 1, wherein said cells having a
phenotype of CD8+/alpha beta TCR-/CD56.sup.bright are predominantly
CD3 epsilon-/CD19+.
4. The cellular composition of claim 1, wherein said hFCs comprise
cells having a phenotype of CD8+/alpha beta TCR-/delta gamma
TCR+/CD3 epsilon+/CD19+.
5. The cellular composition of claim 1, wherein said hFCs comprise
cells having a phenotype of CD8+/alpha beta
TCR-/B220+/CD11c+/CD11b-.
6. The cellular composition of claim 1, wherein about 48% of said
hFCs are CD8+/alpha beta TCR-/CD3 epsilon+, about 33% of said hFCs
are CD8+/alpha beta TCR-/CD19+, about 44% of said hFCs are CD11c+,
about 40% of said hFCs are CD11b+, about 42% of said hFCs are
Foxp3, and about 30% of said hFCs are HLA-DR.
7. The cellular composition of claim 6, wherein about 25% of said
hFCs are CD8+/alpha beta TCR-/IFN-gamma and about 31% of said hFCs
are CD8+/alpha beta TCR-/CXCR4.
8. The cellular composition of claim 1 comprising at least about
40% of the hFCs.
9. The cellular composition of claim 1 comprising at least about
50% of the hFCs.
10. The cellular composition of claim 1 comprising at least about
60% of the hFCs.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S.
application Ser. No. 14/157,888 filed Jan. 17, 2014, which is a
continuation application of U.S. application Ser. No. 12/957,011
filed Nov. 30, 2010, which is a Continuation-In-Part application
of, and claims benefit under 35 U.S.C. .sctn.120 to,
PCT/US09/003340 filed Jun. 1, 2009, which claims benefit under 35
U.S.C. .sctn.119(e) to U.S. Application No. 61/057,724 filed May
30, 2008. This application also claims benefit under 35 U.S.C.
.sctn.119(e) to U.S. Application No. 61/374,460 filed Aug. 17,
2010.
TECHNICAL FIELD
[0002] This invention relates to human facilitating cells, and the
use of such cells in therapeutic protocols.
BACKGROUND
[0003] Facilitating cells (FCs) from mouse have been described.
See, for example, U.S. Pat. No. 5,772,994. FCs are not stem cells,
but significantly improve the initial and long-term engraftment of
stem cells. For example, transplantation of stem cells alone only
prolongs survival of a transplant patient, while the presence of
FCs result in sustained and long-term HSC engraftment. See, also,
Kaufman et al., 2005, J. Exp. Med., 201:373-83).
SUMMARY
[0004] The present invention relates to human facilitating cells
(hFCs), methods of isolating hFCs, and methods of using hFCs for
facilitating reconstitution of a damaged or destroyed hematopoietic
system with stem cells as well as for inducing donor-specific
tolerance for the transplantation of donor cells, tissues and solid
organs.
[0005] In one aspect, a cellular composition comprising is
provided. Such a cellular composition can include at least about
30% (e.g., at least about 40%, at least about 50%, or at least
about 60%) human facilitating cells (hFCs), wherein the hFCs
comprise cells having a phenotype of CD8+ alpha beta
TCR-/CD56d.sup.dim/neg and cells having a phenotype of CD8+/alpha
beta TCR-/CD56.sup.bright.
[0006] In some embodiments, the cells have a phenotype of
CD8+/alpha beta TCR-/CD56.sup.dim/neg are predominantly CD3
epsilon+/CD19-. In some embodiments, the cells have a phenotype of
CD8+/alpha beta TCR-/CD56.sup.bright are predominantly CD3
epsilon-/CD19+. In some embodiments, the hFCs include cells having
a phenotype of CD8+/alpha beta TCR-/delta gamma TCR+/CD3
epsilon+/CD19+. In some embodiments, the hFCs include cells having
a phenotype of CD8+/alpha beta TCR-/B220+/CD11c+/CD11b-. In some
embodiments, about 48% of the hFCs are CD8+/alpha beta TCR-/CD3
epsilon+, about 33% of the hFCs are CD8+/alpha beta TCR-/CD19+,
about 44% of the hFCs are CD11c+, about 40% of the hFCs are CD11b+,
about 42% of the hFCs are Foxp3, and about 30% of the hFCs are
HLA-DR. In some embodiments, about 25% of the hFCs are CD8+/alpha
beta TCR-/IFN-gamma and about 31% of the hFCs are CD8+/alpha beta
TCR-/CXCR4.
[0007] In one aspect, a therapeutic cellular composition is
provided. Such a therapeutic cellular composition can include human
hematopoietic stem cells (HSCs), wherein the HSCs have a phenotype
of CD34+; human facilitating cells (hFCs), wherein the hFCs
comprise cells having a phenotype of CD8+/alpha beta
TCR-/CD56.sup.dim/neg and cells having a phenotype of CD8+/alpha
beta TCR-/CD56.sup.bright; and human alpha beta TCR+ T cells,
wherein the alpha beta TCR+ T cells are present in an amount that
is greater than would be considered therapeutic. In some
embodiments, the therapeutic cellular composition is for delivery
to a recipient. Also in some embodiments, the alpha beta TCR+ T
cells are present in an amount between about 2.0.times.10.sup.6 and
about 5.0.times.10.sup.6 alpha beta TCR+ T cells/kg recipient body
weight. In some embodiments, the number of alpha beta TCR+ T cells
are adjusted to between about 2.0.times.10.sup.6 and about
5.0.times.10.sup.6 alpha beta TCR+ T cells/kg recipient body
weight. In some embodiments, the number of alpha beta TCR+ T cells
are adjusted to between about 3.0.times.10.sup.6 and about
4.2.times.10.sup.6 alpha beta TCR+ T cells/kg recipient body
weight.
[0008] In one aspect, a method of making a therapeutic cellular
composition for delivery to a recipient is provided. Such a method
typically includes providing a donor source of hematopoietic stem
cells (HSCs); depleting alpha beta TCR+ T cells from the donor
source to produce a depleted donor source; adjusting the number of
alpha beta TCR+ T cells in the depleted donor source to greater
than 1.times.10.sup.5 alpha beta TCR+ T cells per kg recipient body
weight, thereby producing a therapeutic cellular composition for
delivery to a recipient. In some embodiments, the source of HSCs is
bone marrow, thymus, peripheral blood, fetal liver, or embryonic
yolk sac. In certain embodiments, the T cells are depleted using
one or more antibodies. In some embodiments, the one or more
antibodies are conjugated to magnetic beads. It is a feature of the
disclosure that the hFCs described herein improve the engraftment
ability of the HSCs compared to HSCs engrafted in the absence of
the hFCs
[0009] In one aspect, a method of making the immune system of a
recipient chimeric with the immune system of a donor is provided.
Such a method typically includes administering the therapeutic
cellular composition described above to the recipient, wherein the
recipient has been conditioned.
[0010] In some embodiments, the conditioning of the recipient
includes a dose of total body irradiation (TBI), wherein the total
body irradiation does not exceed 300 cGy. In some embodiments, the
therapeutic cellular composition is administered to the recipient
intravenously. In some embodiments, the recipient's immune system
is considered to be chimeric with the donor's immune system when
the recipient's immune system is at least about 1% donor
origin.
[0011] In some embodiments, the recipient has a disease. For
example, the disease can be an autoimmune disease, leukemia, a
hemoglobinopathy, an inherited metabolic disorder, or a disease
that necessitates an organ transplant. Representative autoimmune
diseases include diabetes, multiple sclerosis, and systemic lupus
erythematosus. In some embodiments, the disease is an infection by
an immunodeficiency virus or hepatitis. In some embodiments, the
disease can be a hematopoietic malignancy, anemia,
hemoglobinopathies, or an enzyme deficiency. In some embodiments,
the transplanted organ is heart, skin, liver, lung, heart and lung,
kidney, pancreas, or an endocrine organ (e.g., a thyroid gland,
parathyroid gland, a thymus, adrenal cortex, or adrenal
medulla).
[0012] In one aspect, a cellular composition is provided that
includes at least about 30% human facilitating cells (hFCs) having
a phenotype of CD8+/TCR-/CD56.sup.dim/neg. Such a cellular
composition further can include hematopoietic stem cells (HSCs),
wherein the HSCs have a phenotype of CD34+, wherein the HSCs are
MHC-matched with the hFCs. In another aspect, a cellular
composition is provided that includes human hematopoietic stem
cells (HSCs), wherein the HSCs have a phenotype of CD34+; and human
facilitating cells (hFCs), wherein the hFCs have a phenotype of
CD8+/TCR-/CD56.sup.dim/neg. In one embodiment, the hFCs
CD56.sup.dim/neg phenotype is CD56.sup.dim.
[0013] According to this disclosure, hFCs further can have a
phenotype of CD3+/CD16+/CD19+/CD52+. In addition, hFCs can have a
phenotype, without limitation, of CXCR4, CD123, HLADR, NKp30,
NKp44, NKp46, CD11c, and CD162, and hFCs further can be
characterized by the presence of markers such as, without
limitation, CD11a, CD11b, CD62L, and FoxP3. Typically, the hFCs
described herein improve the engraftment ability of the HSCs
compared to HSCs engrafted in the absence of the hFCs. Cellular
compositions as described herein can include at least about 50%
(e.g., 75%, or 90%) of the hFCs. Typically, the phenotype of the
cells is determined by antibody staining or flow cytometry.
[0014] In one aspect, a pharmaceutical composition is provided that
includes a cellular composition as described herein. In another
aspect, methods of treating a human suffering from a disease are
provided. Such methods generally include administering a
pharmaceutical composition that includes a cellular composition as
described herein to a human. In still another aspect, methods of
transplanting donor cells, tissues, or organs into a human
recipient are provided. Such methods generally include
administering a pharmaceutical composition that includes a cellular
composition as described herein to the human recipient.
[0015] Such methods can further include partially conditioning the
human by exposure to total body irradiation, an immunosuppressive
agent, a cytoreduction agent, or combinations thereof prior to the
administration of the pharmaceutical composition. In one
embodiment, the total body irradiation is 200 cGy. A pharmaceutical
composition as described herein can be administered
intravenously.
[0016] In one embodiment, the disease is an autoimmune disease such
as diabetes, multiple sclerosis, or systemic lupus erythematosus.
In another embodiment, the disease is an infection by an
immunodeficiency virus or hepatitis. In yet another embodiment, the
disease is chosen from a hematopoietic malignancy, anemia,
hemoglobinopathies, and an enzyme deficiency.
[0017] Representative donor tissues or organs include, without
limitation, heart, skin, liver, lung, heart and lung, kidney,
pancreas, or an endocrine organ such as a thyroid gland,
parathyroid gland, a thymus, adrenal cortex, or adrenal medulla.
Donor cells can be pancreatic islet cells, neurons, or
myocytes.
[0018] In yet another aspect, methods for obtaining a cellular
composition that includes at least about 0.5% to 8.0% hFCs is
provided. Such a method generally includes providing a
hematopoietic cell composition; and removing cells from the
hematopoietic cell composition that have a phenotype of alpha beta
TCR- and gamma delta TCR-. Such methods can further include
selecting for cells that have a phenotype of CD8+; selecting for
cells that have a phenotype of CD56.sup.dim/neg; and selecting for
cells that have a phenotype of CD3+/CD16+/CD19+/CD52+. In addition,
such methods can further include selecting for cells that have a
phenotype of CXCR4, CD123, HLADR, NKp30, NKp44, NKp46, CD 11 c, and
CD162, and such methods can still further include selecting for
cells that have a phenotype of, wiithout limitation, CD11a, CD11b,
CD62L, and FoxP3. A cellular composition produced by such methods
also can include CD34+ hematopoietic stem cells (HSCs).
[0019] Cells can be removed and/or selected for using an antibody
(e.g., an antibody conjugated to a magnetic bead). In addition or
alternatively, the hematopoietic cell composition can be separated
by density gradient centrifugation to obtain cells in the
mononuclear cell fraction. In one embodiment, the hematopoietic
cell composition is contacted with a growth factor. The
hematopoietic cell composition can be obtained from bone marrow,
thymus, peripheral blood, fetal liver, or embryonic yolk sac. In
one embodiment, cellular composition includes at least about 50%
hFCs (e.g., at least about 75% hFCs).
[0020] Unless otherwise defined, 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 technology belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present methods and compositions, suitable methods and materials
are described below. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their
entirety.
[0021] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages will be apparent from the
drawings and detailed description, and from the claims.
DESCRIPTION OF DRAWINGS
[0022] FIG. 1 are graphs showing representative phenotypic analysis
of hFCs.
[0023] FIG. 2 is a graph showing the CD56.sup.dim/neg and
CD56.sup.bright sub-populations within CD8+/alpha beta
TCR-hFCs.
[0024] FIG. 3A is a graph showing representative phenotypic
analysis of the CD8+/alpha beta TCR-/CD56.sup.dim/neg hFC
sub-population, and FIG. 3B is a graph showing representative
phenotypic analysis of the CD8+/alpha beta TCR-/CD56.sup.bright hFC
sub-population.
[0025] FIG. 4 shows the gating strategy for sorting and enumeration
of human HSCs.
[0026] FIG. 5 shows the gating strategy for sorting and enumerating
human hFCs.
[0027] FIG. 6A is a graph showing the results of immunological
monitoring in response to a number of stimulators 1-month
post-transplantation in transplant patient SCD#3, and FIG. 6B is a
graph showing the absolute neutrophil count (ANC) in transplant
patient SCD#4.
[0028] FIG. 7A is a graph showing the chimerism of transplant
patient SCD #3, and FIG. 7B is a graph showing the source of
hemoglobin from the same transplant patient.
[0029] FIG. 8 are graphs showing the chimerism (Panel A), the
source of hemoglobin (Panel B), and the reticulocyte counts (Panel
C) for transplant patient SCD #4.
[0030] FIG. 9 are graphs showing the amount of the ANC and white
blood cells (WBCs) (Panel A), the platelet count (Panel B), and the
percent chimerism (Panel C) in a patient following solid organ
transplant.
[0031] FIG. 10A is a graph showing the platelet count and FIG. 10B
is a graph showing the percent chimerism in patient #2 following
solid organ transplant.
[0032] FIG. 11 are graphs showing the ANC (Panel A), the recovery
of B cells, CD4+ cells, and CD8+ cells (Panel B), and the platelet
count (Panel C) following solid organ transplant in patient
#12.
[0033] FIG. 12 is a schematic showing a general nonmyeloablative
conditioning and post-transplant immunosuppression regimen as
exemplified herein.
[0034] FIG. 13 are graphs showing the chimerism (Panel A),
multilineage chimerism (Panel B), response to a number of
stimulators (Panel C), creatinine levels (Panel D), platelet count
(Panel E), and white blood count and ANC (Panel F) in living donor
kidney transplant Subject #3.
DETAILED DESCRIPTION
[0035] Human hFCs (hFCs) are provided that facilitate initial stem
cell engraftment and that are required for sustained HSC
engraftment. Also provided are methods of purifying such hFCs from
bone marrow or other physiological sources of hematopoietic cells.
A variety of separation procedures are provided, which generally
are based on the presence or absence of specific markers as
disclosed herein.
Human Facilitating Cells (hFCs), Cellular Compositions Containing
hFCs, and Methods of Making
[0036] Human facilitating cells (hFCs) have been identified and are
described herein. hFCs are generally characterized as CD8+ and
alpha beta TCR-. hFCs also can be gamma delta TCR- or gamma delta
TCR+ (i.e., the absence of gamma delta TCR cells is not required).
The CD8+/alpha beta TCR-hFCs can be characterized by the presence
of cells expressing the following markers: CD3 epsilon (expressed
by about 48% of hFCs), CD19 (expressed by about 33% of hFCs), CD11c
(expressed by about 44% of hFCs), CD11b (expressed by about 40% of
hFCs), Foxp3 (expressed by about 42% of hFCs), HLA-DR (expressed by
about 30% of hFCs), and CD123 (expressed by about 8% of hFCs) (FIG.
1A). hFCs also can be characterized by the presence of cells
expressing IFN-gamma (about 25% of hFCs) and CXCR4 (about 31% of
hFCs) (FIG. 1B). In addition, about 65% of hFCs resemble
tolerogenic plasmacytoid dendritic cells (B220+/CD11c+/CD11b-), and
hFCs are capable of inducing antigen-specific T.sub.reg cells.
Further, hFCs can be characterized by the presence, in lower
levels, of markers such as, without limitation, CD16, CD52, NKp30,
NKp44, NKp46, CD162, CD11a and CD62L.
[0037] Within the population of CD8+/alpha beta TCR-hFCs, there are
two subpopulations: CD8+/alpha beta TCR-/CD56.sup.dim/neg (about
55% of hFCs) and CD8+/alpha beta TCR-/CD56.sup.bright (about 45% of
hFCs) (FIG. 2). As is understood by those skilled in this art,
CD56.sup.dim/neg cells refer to a population of cells that express
a relatively small amount of CD56 (CD56.sup.dim) and cells that do
not express CD56 (CD56.sup.neg); while CD56.sup.bright cells refer
to cells that express a relatively large amount of CD56
(CD56.sup.bright).
[0038] Within the CD8+/alpha beta TCR-/CD56.sup.dim/neg
sub-population of hFCs, the majority of cells express CD3 epsilon
(about 80%), approximately a third of the cells express HLA-DR
(about 30%), and a lower percentage of cells express CD11c (about
17%), CD19 (about 16%), CD11b (about 14%), and CD123 (about 11%)
(FIG. 3A). Thus, the majority of cells within the CD8+/alpha beta
TCR-/CD56.sup.dim/neg hFC sub-population are CD3 epsilon+/CD19-.
Within the CD8+/alpha beta TCR-/CD56.sup.bright sub-population of
hFCs, approximately 65% of cells express CD11c, about 67% of the
cells express CD11b, and about 40% of the cells express HLA-DR,
while CD3 epsilon, CD19, and CD123 are expressed at much lower
levels (about 29%, about 25%, and about 10%, respectively) in this
sub-population (FIG. 3B). Thus, the majority of cells within the
CD8+/alpha beta TCR-/CD56.sup.bright hFC sub-population are CD3
epsilon-/CD19+.
[0039] hFCs can be obtained from bone marrow, or any other
physiologic source of hematopoietic cells such as, without
limitation, the spleen, thymus, blood, embryonic yolk sac, or fetal
liver. In one embodiment, hFCs are obtained from mobilized
peripheral blood (in the presence of, for example, granulocyte
colony-stimulating factor (G-CSF) or granulocyte-macrophage colony
stimulating factor (GM-CSF). In another embodiment, hFCs are
obtained from vertebral bone marrow.
[0040] Once hematopoietic cells are obtained, hFCs can be enriched,
purified (or substantially purified) by various methods that
typically use antibodies that specifically bind particular markers
to select those cells possessing (or lacking) those particular
markers. Cell separation techniques include, for example, cell
sorting using a fluorescence activated cell sorter (FACS) and
specific fluorochromes; biotin-avidin or biotin-streptavidin
separations using biotin conjugated to cell surface marker-specific
antibodies and avidin or streptavidin bound to a solid support
(e.g., affinity column matrix or a plastic surface); magnetic
separations using antibody-coated magnetic beads; or destructive
separations such as antibody and complement or antibody bound to
cytotoxins or radioactive isotopes. Methods of making antibodies
that can be used in cell separations are well known in the art.
See, for example, U.S. Pat. No. 6,013,519.
[0041] Separation using antibodies directed toward specific markers
can be based upon negative or positive selections. In separations
based on negative selection, antibodies that are specific for
markers that are present on undesired cells (non-hFCs) and that are
not present on the desired cells (hFCs) are used. Those (undesired)
cells bound by the antibody are removed or lysed and the unbound
cells retained. In separations based on positive selection,
antibodies that are specific for markers that are present on the
desired cells (hFCs) are used. Those cells bound by the antibody
are retained. It will be understood that positive and negative
selection separations may be used concurrently or sequentially. It
will also be understood that the present disclosure encompasses any
separation technique that can be used to enrich or purify the hFCs
described herein.
[0042] One well-known technique for antibody-based separation is
cell sorting using, for example, a FACS. Briefly, a suspended
mixture of hematopoietic cells are centrifuged and resuspended in
media. Antibodies that are conjugated to fluorochromes are added to
allow the binding of the antibodies to the specific cell surface
markers. The cell mixture is then washed and run through a FACS,
which separates the cells based on their fluorescence, which is
dictated by the specific antibody-marker binding. Separation
techniques other than cell sorting additionally or alternatively
can be used to obtain hFCs. One such method is biotin-avidin (or
streptavidin)-based separation using affinity chromatography.
Typically, such a technique is performed by incubating
hematopoietic cells with biotin-coupled antibodies that bind to
specific markers, followed by passage of the cells through an
avidin column. Biotin-antibody-cell complexes bind to the column
via the biotin-avidin interaction, while non-complexed cells pass
through. The column-bound cells can be released by perturbation or
other known methods. The specificity of the biotin-avidin system is
well-suited for rapid separation.
[0043] Cell sorting and biotin-avidin techniques provide highly
specific means for cell separation. If desired, less specific
separations can be utilized to remove portions of non-hFCs from the
hematopoietic cell source. For example, magnetic bead separations
can be used to initially remove non-facilitating differentiated
hematopoietic cell populations including, but not limited to,
T-cells, B-cells, natural killer (NK) cells, and macrophages (MAC)
as well as minor cell populations including megakaryocytes, mast
cells, eosinophils, and basophils. In addition, cells can be
separated using density-gradient separation. Briefly, hematopoietic
cells can be placed in a density gradient prepared with, for
example, Ficoll or Percoll or Eurocollins media. The separation can
then be performed by centrifugation or automatically with, for
example, a Cobel & Cell Separator '2991 (Cobev, Lakewood,
Colo.). Additional separation procedures may be desirable depending
on the source of the hematopoietic cell mixture and its content.
For example, if blood is used as a source of hematopoietic cells,
it may be desirable to lyse red blood cells prior to the separation
of any fraction.
[0044] Although separations based on specific markers are
disclosed, it will be understood that the present disclosure
encompasses any separation technique(s) that result in a cellular
composition that is enriched for hFCs, whether that separation is a
negative separation, a positive separation, or a combination of
negative and positive separations, and whether that separation uses
cell sorting or some other technique, such as, for example,
antibody plus complement treatment, column separations, panning,
biotin-avidin technology, density gradient centrifugation, or other
techniques known to those skilled in the art. Most sources of
hematopoietic cells naturally contain about 0.5% to about 8% (e.g.,
typically about 1%) hFCs. The separations such as those disclosed
herein can yield cellular compositions that are enriched for hFCs
(i.e., include a greater number of hFCs than are found naturally in
physiological hematopoietic cell sources). For example, cellular
compositions are provided in which at least about 5% (e.g., at
least about 8%, 10%, 12%, 15%, 20% or more) of the cells are hFCs
as described herein. These compositions are referred to as
"enriched" for hFCs. In another example, cellular composition are
provided in which at least about 30% (e.g., at least about 35%,
40%, 50% or more) of the cells are hFCs as described herein. The
compositions are referred to as "purified" for hFCs. Further
processing, by either or both positive or negative selections, can
yield cellular compositions in which at least about 60% of the
cells (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, or 99%)
are hFCs as described herein.
[0045] Exemplary methods of obtaining cellular compositions that
include hFCs are described herein. Those skilled in this art would
understand that the examples described herein can be modified in a
number of ways to still obtain hFCs or to obtain different amounts
of hFCs. In the following examples, bone marrow is the source of
hematopoietic stem cells. Bone marrow can be harvested (e.g., from
a donor) by various methods well known to those skilled in the art.
For example, bone marrow can be harvested from the long bones
(e.g., femora or tibia), but also can be obtained from other bone
cavities or the spine.
[0046] In one exemplary method, non-hFCs and non-HSCs can be
removed from the bone marrow using one or more negative selections
described herein. For example, T cells, also known as graft vs.
host disease (GVHD)-producing cells, can be specifically removed
from the cellular composition using antibodies directed toward T
cell-specific markers such as alpha beta TCR+. In certain
embodiments, an antibody directed toward delta gamma TCR+ can be
used to remove a further subset of T cells. The resulting cellular
composition is enriched for hFCs and HSCs, and also will contain
other immature progenitor cells such as immature lymphoid and
myeloid progenitor cells. Representative cellular compositions
enriched for hFCs were deposited with the American Type Culture
Collection (ATCC: Manassas, Va.) on ______ and assigned Accession
Nos. ______. These deposits were made for reference purposes only
and were not made for purposes related to 35 U.S.C. .sctn.112.
[0047] In another exemplary method, hFCs can be obtained from the
bone marrow using one or more positive selections described herein.
For example, hFCs can be purified by cell sorting (e.g., using
FACS) with one or more of the markers described herein (e.g., CD8+,
CD19, CD56).
[0048] In certain instances (e.g., non-therapeutic), it may be
desirable to remove the HSCs from the cellular composition. HSCs
can be removed from bone marrow using, for example, antibodies that
bind CD34+ and, optionally, CD45+. See, for example, U.S. Pat. No.
5,061,620 or the LC Laboratory Cell Separation System, CD34 Kit
(CellPro, Inc., Bothell, Wash.).
Methods of Using hFCs and Cellular Compositions Containing hFCs
[0049] The ability of hFCs to enhance engraftment of donor bone
marrow cells in a recipient indicates that hFCs are useful in
facilitating various therapy protocols. Using a cellular
composition that is enriched for hFCs (e.g., contains about 5% to
about 12% hFCs) significantly improves durable engraftment and
eliminates graft vs. host disease (GVHD). Although not bound by any
particular mechanism, it is believed that, once administered, the
hFCs home to various hematopoietic cell sites in the recipient's
body, including bone cavity, spleen, fetal or adult liver, and
thymus. The hFCs become seeded at the proper sites, engraft, and
begin establishing a chimeric immune system. It is possible that
both the stem cells and the hFCs complex together to seed the
appropriate site for engraftment.
[0050] Methods of administering a therapeutic cellular composition
comprising hFCs to a recipient also are described herein. A
therapeutic cellular composition as used herein refers to a
composition that includes hFCs and HSCs. Such a composition can be
produced using any of the methods described herein (e.g., positive
and/or negative selections). A therapeutic cellular composition for
administration to a recipient may include a total of between about
1.times.10.sup.8 cells and 3.times.10.sup.8 cells per kilogram of
dosing weight of the recipient. Within a therapeutic cellular
composition, the number of HSCs can be between about
1.times.10.sup.5 and 18.times.10.sup.6 HSCs per kg of recipient
dosing weight, and a similar range of hFCs can be administered. The
exact numbers of cells that are used, however, will depend on many
factors, including the number of cells in the original source of
hematopoietic stem cells, the number of cells (e.g., hFCs and/or
HSC) present after processing (e.g., enrichment and/or
purification), as well as the condition of the recipient's
health.
[0051] As described herein, obtaining hFCs typically involves
depleting the alpha beta TCR+ T cells, as these are considered
GVHD-producing cells. Therapeutically, however, the presence of
alpha beta TCR+ T cells has been found to be beneficial in the
cellular composition of HSCs and hFCs. As shown in the Example
section herein, a cellular composition that includes alpha beta
TCR+ T cells at a level that is greater than is generally
considered to be therapeutic surprisingly promoted chimerism and
engraftment. It is generally accepted that about 1.times.10.sup.5
alpha beta TCR+ T cells/kg of recipient body weight is considered a
lethal amount of T cells. However, in the methods described herein,
amounts greater than that were routinely administered to recipients
without adverse effects. Specifically, amounts between about
2.0.times.10.sup.6 and 5.0.times.10.sup.6 alpha beta TCR+ T cells
(e.g., between about 2.5.times.10.sup.6 and 4.5.times.10.sup.6
alpha beta TCR+ T cells/kg recipient body weight; between about
3.0.times.10.sup.6 and 4.0.times.10.sup.6 alpha beta TCR+ T
cells/kg recipient body weight; about 3.0.times.10.sup.6 and
4.2.times.10.sup.6 alpha beta TCR+ T cells/kg recipient body
weight; about 3.2.times.10.sup.6 alpha beta TCR+T cells/kg
recipient body weight; or about 3.8.times.10.sup.6 alpha beta TCR+
T cells/kg recipient body weight) can be included in a therapeutic
cellular composition.
[0052] Accordingly, depending on the procedures and methods used to
obtain the HSC and hFC therapeutic cellular composition, the number
of alpha beta TCR+ T cells in the composition may need to be
adjusted. For example, in certain embodiments, alpha beta TCR+ T
cells can be added back to the T cell-depleted HSC and hFC
composition in order to obtain the desired number. In other
embodiments, the depletion step can be modified so that only the
desired about of T cells are depleted, thereby leaving the desired
amount of T cells in the composition. In order to achieve the
desired amount of T cells in a therapeutic cellular composition,
the number of T cells can be determined, for example, prior to
depletion (e.g., in the starting material) or following the
depletion step. Methods of determining the number of cells (e.g., T
cells) in a sample are well known in the art (e.g., FACS).
[0053] Therapeutic cellular compositions generally are administered
intravenously, but other modes of administration such as direct
bone injection can be used. The therapeutic cellular compositions
described herein, even in the presence of higher-than-therapeutic
levels of alpha beta TCR+ T cells, result in durable chimerism. As
used herein, durable chimerism refers to a recipient's immune
system that is at least about 1% (e.g., at least about 2%, 3%, 4%,
5%, 6%, 7%, 8%, 9%, 10%, 25%, 50%, 75% or more (e.g., 100%)) donor
origin for greater than 6-months post-transplant (e.g., 1-year or
more post-transplant). In addition, durable chimerism can be
achieved using the therapeutic cellular composition described
herein even in recipients who are not HLA-matched to their donor or
who are only partially matched with their donor. Accordingly, the
therapeutic cellular compositions described herein allow for
transplantation between a donor and a recipient that are syngeneic
to one another and should allow for transplantation between a donor
and a recipient that are allogeneic to one another.
[0054] Traditionally, methods of establishing a chimeric immune
system required destroying the immune system of the recipient,
which results in ablation of the recipient's HSCs. This may be
accomplished by techniques well known to those skilled in the art
and include, without limitation, irradiating the recipient with
selected levels of total body irradiation, administering specific
toxins or chemotherapeutic agents to the recipient, administering
specific monoclonal antibodies or monoclonal antibodies attached to
toxins or radioactive isotopes to the recipient, or combinations
thereof. Notably, administering the hFCs described herein (e.g., in
the therapeutic cellular composition) to a recipient significantly
reduces the amount conditioning required of a recipient for
successful engraftment and also significantly reduces the amount of
immunosuppression required following transplantation. For example,
destroying a recipient's immune system often involves lethally
irradiating the recipient with 950 centigray (cGy) of total body
irradiation (TBI), while the procedures described herein utilize a
conditioning regimen with as little as 25 cGy to 200 cGy of
TBI.
[0055] The ability to establish successful chimerism allows for
significantly improved survival following transplant. The present
disclosure provides for methods of transplanting a donor
physiological component, such as, for example, organs, tissue, or
cells. Using the hFCs in the methods disclosed herein results in a
recipient who has a chimeric immune system, which is completely
immunotolerant to transplanted donor organ, tissue, or cells, but
competently rejects third party grafts. Transplanted donor organ,
tissue, or cells are able to perform their respective functions in
the recipient. For example, transplanted islet cells can provide an
effective treatment for diabetes. In addition, permanent acceptance
of endocrine tissue grafts (thyroid, parathyroid, adrenal cortex,
adrenal medulla, islets) as well as kidney, liver, heart, and
composite tissues such as face, hand and other extremities has been
demonstrated. It will be understood that a mixed chimeric immune
system can be produced in a recipient before, during, or after
transplantation of an organ, tissue or cells, but typically is
produced before or at the same time as the transplantation.
[0056] The use of hFCs in establishing a chimeric immune system can
significantly expand the scope of diseases that can be treated
using bone marrow transplantation. Beyond transplantation (e.g.,
heart, kidney, liver, pancreatic islets, and hand or face), the
ability to establish a successful chimeric hematopoietic system in
a recipient can be used to treat other diseases or disorders that
are not currently treated by bone marrow transplantation because of
the morbidity and mortality associated with GHVD. Autoimmune
diseases involve attack of an organ or tissue by one's own immune
system. However, when a chimeric immune system is established, the
body can relearn what is foreign and what is self. Establishing a
chimeric immune system using the hFCs described herein can reduce
or halt the autoimmune attack causing the condition. Autoimmune
diseases that can be treated using the hFCs described herein
include, for example, type I diabetes, systemic lupus
erythematosus, multiple sclerosis, rheumatoid arthritis, psoriasis,
or Crohn's colitis.
[0057] It may also be possible to treat Alzheimer's disease using
the cellular compositions described herein. The cellular
compositions disclosed herein also can be used to treat
hemoglobinopathies such as, for example, sickle cell anemia,
spherocytosis or thalassemia, as well as metabolic disorders such
as Hunters disease, Hurlers disease, chronic granulomatous disease,
leukodystrophy, and enzyme defects. In addition, the cellular
compositions described herein can be used to treat leukemias or
other rare childhood disorders (e.g., ADA deficiency, aplastic
anemia or SCID), or the cellular compositions described herein can
be used in regenerative repair (e.g., macular degeneration,
myocardial infarction, or islet regeneration).
[0058] In accordance with the present disclosure, there may be
employed conventional molecular biology, cell biology, microbiology
and biochemical techniques within the skill of the art. Such
techniques are explained fully in the literature. The methods and
compositions will be further described in the following examples,
which do not limit the scope of the methods and compositions
described in the claims.
EXAMPLES
Section A--Colony Forming Cell Assays
Example 1
Purification of HSC and hFC
[0059] HSC and hFC were isolated from Human Vertebral Bone Marrow
(VBM) or Mobilized Peripheral Blood (MPB) by multiparameter, live
sterile cell sorting (FACSVantage SE: Becton Dickinson). Briefly,
VBM or MPB was stained with directly labeled monoclonal antibodies
(mAbs) at saturating concentrations for 30 min. HSCs: CD34+/CD45+;
and hFCs: CD8+/TCR-/CD56.sup.dim/neg. Both cell populations were
sorted and analyzed for purity. Only 85% or greater purity levels
were accepted.
Example 2
HSC and hFC Sorting and Enumeration
[0060] HSCs were sorted and enumerated based on the ISHAGE
protocol. See, Sutherland et al., 1996, "The ISHAGE guidelines for
CD34+ cell determination by flow cytometry," J. Hematotherapy,
5:213-26. Briefly, CD45-FITC/CD34-PE combination parameters
provided a clinically relevant reflection of the peripheral blood
stem/progenitor cell compartment. Plot 1 was formatted with Forward
Scatter (FSC; x-axis) vs Side Scatter (SSC; y-axis), and a region
(R1) was drawn around the lymphocyte, monocyte and granulocyte
populations excluding debris. From R1, Plot 2 was formatted with
CD45 FITC versus Side Scatter, and R1 was drawn so that CD45-
events were excluded. From R2, Plot 3 was formatted with CD34 PE
versus Side Scatter, and R3 was drawn only around the CD34+
population. From R3, Plot 4 was formatted with CD45-FITC versus SSC
of CD34+ cells. Cells forming a cluster with characteristic low SSC
and low to intermediate CD45 fluorescence were gated and designated
R4. Nonspecific stained events were excluded from this region. From
R4, Plot 5 was formatted with FSC (x-axis) versus SSC (y-axis). A
cluster of events meeting all the fluorescence and light scatter
criteria of CD34+ stem/progenitor cells appeared in Plot 5.
[0061] FIG. 4 shows the gating strategy for the sorting and
enumeration of human HSCs using the ISHAGE protocol. FIG. 5 shows
the gating strategy for the sorting and enumeration of human
hFCs.
Example 3
Colony-Forming Cell Assay with hFCs
[0062] Following cell sorting, HSCs (CD45+/CD34+) alone or HSCs and
hFCs (CD45+/CD34+ plus CD8+/TCR-/CD56.sup.dim/neg) or HSCs+ T cells
as a control were either immediately plated in methylcellulose (0
hr) or pre-incubated for 18 hrs in cell culture media before
plating in methylcellulose. All cell samples were cultured in
quadruplicate. After 14 days of culturing at 37.degree. C. and 5%
CO2, colonies containing more than 50 cells were scored.
[0063] Without pre-incubation, there was no significant difference
in colonies generated by HSC alone vs. HSC plus hFC. Strikingly,
when HSCs were co-incubated with hFCs for 18 hrs prior to placement
in the CFC assay, hFC significantly (p<0.005) enhanced colony
formation compared to HSC alone and HSC co-incubated with CD8+ T
cells. These results indicate that human hFCs, like mouse hFCs,
exert a protective effect on HSCs and promote the generation of
more primitive multipotent progenitors in vitro.
Example 4
Colony-Forming Cell Assay with a Sub-Population of hFCs
[0064] Colony Forming Culture (CFC) Assay: 15,000 HSCs were
cultured with or without 30,000 CD8+/alpha beta
TCR-/CD56.sup.dim/neg hFCs for 0 hrs or 18 hrs in culture media in
a 96 well plate and incubated at 37.degree. C. After culture, cells
were resuspended in methylcellulose and used in a CFC Assay.
Colonies were counted on day 14.
[0065] Summary and Results: To evaluate the function of CD8+/alpha
beta TCR-/CD56.sup.dim/neg hFCs in vitro, HSCs were incubated with
CD8+/alpha beta TCR-/CD56.sup.dim/neg hFCs for 18 hrs and then
cultured in methylcellulose for 14 days in a colony-forming cell
assay. HSC plus CD8+/alpha beta TCR-/CD56.sup.dim/neg hFCs
generated significantly more colonies compared with HSCs alone
(p=0.0038), demonstrating that CD8+/alpha beta
TCR-/CD56.sup.dim/neg hFCs have a direct effect on the
clonogenicity of HSCs.
Section B--Characterization of Human hFCs In Vivo
Example 1
Chimerism and Engraftment in a Mouse Model
[0066] It has been shown previously that CD8.sup.+/TCR.sup.-hFCs
enhance engraftment of purified HSCs in allogeneic and syngeneic
mouse recipients (Fugier et al., 2005, J. Exp. Med.,
201(3):373-383). In addition, it has been shown in mice that hFCs
enhance the clonogenicity and promote the generation of more
primitive multipotent HSC progenitors in vitro (Rezzoug et al.,
2008, J. Immunology, 180(1):49-57).
[0067] One goal was to achieve human HSC chimerism in a mouse
model. Briefly, CD34.sup.+, CD45.sup.+ human HSCs were sorted from
G-CSF mobilized peripheral blood, and 100,000 sorted human HSCs
were transplanted into NOD/SCID/IL2 receptor (IL2R) .gamma.
chain.sup.null mice conditioned with 325 cGy TBI. Whole blood was
collected from transplanted mice one month following
transplantation, and PBL typing was performed using antibodies
specific for human T cells, B cells, natural killer cells,
dendritic cells, and monocytes. Results showed that an average of
3.2% human HSC chimerism was achieved following transplantation
with 100,000 hHSCs.
[0068] Experiments then were performed in which 100,000 hHSCs alone
or 100,000 hHSCs+300,000 hFCs were transplanted into
NOD/SCID/IL2R.gamma..sup.null mice conditioned with 325 cGy TBI.
Multilineage PBL typing was performed at 30 days after
transplantation as described above.
[0069] The results of these experiments demonstrated that the
HSC+hFC group produced a higher percentage of human T cells (CD4,
CD8, DC; see Table 1) and human monocytes (CD33; Table 2) compared
to the HSC alone group. The percentage of donor chimerism in
lymphoid gate and myeloid gate are summarized in Table 3.
TABLE-US-00001 TABLE 1 Percentage of human T cells, NK cells, B
cells, and DCs in lymphoid gate T cells .alpha..beta./ B
.delta..gamma. NK cell DC Group Mouse CD8 CD4 CD3 TCR CD56 CD19
CD11c HSC alone A 0 0.1 0.1 0.1 0.1 0.1 0 B 0 0 0.1 0.3 0.2 0 0 C 0
0 0 0.1 0 0 0 HSC + D 0.1 0.5 0.4 0.5 0.1 0.1 0.1 hFC E 0.1 0.1 0.1
0.1 0.1 0 0.1 F 0 0.1 0.1 0.1 0.1 0.1 0.1
TABLE-US-00002 TABLE 2 Percentage of human DCs and monocytes in
myeloid gate Group Mouse CD11c CD33 HSC alone A 4.8 9.2 B 0.6 5 C 0
0 HSC + hFC D 3.2 6.6 E 4.1 8.1 F 8.2 14.9
TABLE-US-00003 TABLE 3 Percentage of human hematopoietic cells
Lymphoid Myeloid Group Mouse CD45 CD45 HSC alone A 1.5 9.2 B 1.5
5.8 C 0.3 0.3 HSC + hFC D 1.1 8.2 E 0.8 9.4 F 1.2 15.9
Example 2
Engraftment of CD8+/Alpha Beta TCR-/CD56.sup.dim/neg in a mouse
model
[0070] Animals: Five to 6-week-old male non-obese diabetic
(NOD)/SCID/interleukin-2 receptor (IL-2r) gamma-chain knockout
(NSG) mice were purchased from the Jackson Laboratory (Bar Harbor,
Me.).
[0071] Purification of HSCs and hFCs: HSCs and FCs were sorted from
human G-CSF-mobilized peripheral blood by multiparamter, live
sterile cell sorting (FACSVantage SE and FACSAria; Becton
Dickinson, Mountain View, Calif.).
[0072] Phenotype of human CD8+/alpha beta TCR-hFCs: G-CSF mobilized
PBMC were stained with anti-human CD8 alpha, alpha beta TCR, delta
gamma TCR, CD56, CD3 epsilon, CD19, CD11c, CD11b, HLA-DR, Foxp3,
INF-gamma, TGF-beta, CXCR4, and SDF-1 monoclonal antibodies, and
analyzed by LSR using Cell Quest Software (Becton Dickinson).
[0073] HSC and FC transplantation: In the human HSC+FC xenogeneic
model, 100,000 human HSCs with or without 300,000 sorted CD8+/alpha
beta TCR-/CD56.sup.dim/neg hFCs were transplanted into
NOD/SCID/IL-2r gamma.sup.null mice recipients conditioned with 325
cGy TBI.
[0074] Assessment of chimerism: Donor cell engraftment was
evaluated in peripheral blood lymphocytes, bone marrow cells and
splenocytes using 7-color flow cytometry.
[0075] Summary: To evaluate whether human CD8+/alpha beta
TCR-/CD56.sup.dim/net hFCs enhance engraftment of human HSCs in
vivo, 100,000 HSC alone or plus 300,000 CD8+/alpha beta
TCR-/CD56.sup.dim/net hFCs was transplanted into
NOD/SCID/IL2rgn.sup.null (NSG) recipient mice conditioned with 325
cGy of total body irradiation. At 30 days after transplantation, 8
of 21 (38%) recipients of HSC alone engrafted. In contrast, 81% of
recipients (n=16) receiving HSC plus CD8+/alpha beta
TCR-/CD56.sup.dim/net hFCs engrafted, and donor lymphocyte and
donor monocyte chimerism in peripheral blood was 0.53%.+-.0.16% and
3.93%.+-.1.28%, respectively.
[0076] At 6 months after transplantation, NSG recipients of HSC
alone lost donor chimerism in peripheral blood and little to no
donor cells were detected in spleen and bone marrow. In contrast,
NSG recipients of HSC +CD8+/alpha beta TCR-/CD56.sup.dim/net hFCs
exhibited durable donor chimerism in peripheral blood and showed
significantly higher levels of donor chimerism in spleen (about
three-times as many donor lymphocytes and about twice as many donor
monocytes) and bone marrow (about ten-times as many donor
lymphocytes and about four-times as many donor monocytes) compared
to recipients of HSC alone.
Section C--Treatment of Sickle Cell Disease (SCD) in Humans
Example 1
The Sickle Cell Disease (SCD) Preliminary Experiment
[0077] Two sickle-cell disease (SCD) patients were previously
treated in a pilot experiment to try to establish mixed chimerism.
Both SCD patients were at high risk for complications from their
disease. Whether a combination of 200 cGy TBI with fludarabine,
MMF, and CyA could establish engraftment in patients with SCD was
evaluated. Only transient engraftment, however, was achieved. The
conditioning was well tolerated, and no severe adverse events
occurred; however, endogenous hematopoiesis reappeared.
[0078] To overcome the transfusion/sensitization barrier,
improvements were made to the protocol. For example, Campath, which
is a humanized anti-CD52 monoclonal antibody that is a powerful
lytic agent for mature T cells, B cells and NK cells, was added to
the clinical conditioning regimen. Two cycles of Campath were
administered (month-2 and month-1) with the rationale that the
first cycle would deplete mature B cells and cause homeostatic
proliferation of memory B cells to replace the depleted B cells and
the second cycle would deplete the proliferating memory B cells. It
was hypothesized that the broad lymphoid specificity of Campath
would provide a powerful approach to target T and B cells in the
recipient that mediate the alloreactivity induced by transfusion
therapy.
[0079] In one example, four doses of 10 mg/day of Campath-1H were
administered at day-53 to -50, and another four doses of 7 mg/day
of Campath-1H were administered at day-24 to -21. 30 mg/m.sup.2 of
Fludarabine was administered at day-5 to -3, and 200 cGy total body
irradiation was administered at day-1 along with Mycophenolate
mofetil and cyclosporine, which was continued until durable
engraftment. FCs+HSCs were transplanted at day 0.
[0080] Two subjects with SCD have been successfully transplanted
under the revised protocol. Both subjects have maintained
engraftment at 27 and 24 months post-transplant and are
asymptomatic and transfusion independent. It was demonstrated that
mixed chimerism can be established with minimal toxicity in
sensitized recipients through partial recipient conditioning
followed by transplantation with HSCs and hFCs to reduce the risk
of GVHD while preserving engraftment. The reduced-intensity
conditioning approach described herein is safe, well-tolerated and,
in combination with the HSC+hFC graft, sufficient to induce stable
mixed chimerism and dominantly normal RBC production in transfused
patients. Immunocompetence to respond to PHA, Candida, and
alloantigen returned by 1 month post-transplant (FIG. 6A; a
stimulation index of >3 is positive (horizontal line on graph)).
The nadir occurred between day 9 and day 24 for both patients
(absolute neutrophil count [ANC]<1,000) (FIG. 6B).
Example 2
SCD Patient #3--Transplanted in November 2005
[0081] SCD#3 (Date of Birth 2-11-98) is an African American female
who experienced multiple pain crises and episodes of acute chest
syndrome. She was maintained on transfusion therapy. Her
HLA-identical sister with sickle cell trait served as her donor.
The patient was conditioned with four doses of Campath-1H (30
mg/day) starting at day-53, and a second round of four doses of
Campath-1H (30 mg/day) starting at day-24. She received 3 doses of
fludarabine (30 mg/m.sup.2 IV) starting at day-4, and then 200 cGy
of TBI on day 0.
[0082] Post-transplant, she was treated with cyclosporine (1.5
mg/kg/bid) and MMF for 22 months. The immunosuppression was
subsequently tapered and has been discontinued completely. The
patient received 14.1.times.10.sup.6 CD34+ cells/kg body weight,
43.5.times.10.sup.6 alpha beta TCR cells/kg body weight and
5.4.times.10.sup.6 hFCs/kg body weight. She showed 5% donor cell
chimerism on Day 17 and 78% donor cell chimerism on day 32. She has
been asymptomatic and has not required transfusions post
transplant. At day 727 post-transplantation, she was 21% donor cell
chimeric (FIG. 7A), and she had no evidence of GVHD. Although her
total donor chimerism was approximately 30%, she was producing
nearly 100% donor-derived trait RBC (FIG. 7B). At 1259 days
post-transplantation, she produced 100% donor RBC and had T, B, and
myeloid chimerism ranging between 10-30%. She was still transfusion
independent and had not had any complications from her SCD.
[0083] During the processing procedure for SCD #3, some difficulty
was experienced in recovering the correct fraction in the cell
separation (Percoll) procedure due to the density of SCD trait
marrow cells. Because the donor/recipient pair were HLA matched,
the decision was made to abort the process and not deplete the
product. Therefore, the patient received whole bone marrow.
However, the efficacy of the conditioning was established in this
candidate.
Example 3
SCD Patient #4--Transplanted in March 2006
[0084] SCD#4 (Date of birth May 23, 1996) is a Nigerian male who
suffered multiple pain crises and two acute chest syndromes prior
to starting red cell exchange in 1999. The patient's HLA-identical
sibling who had sickle cell trait served as his donor. The patient
received the same non-myeloablative conditioning as SCD #3. His
HSC+hFC dose was 5.24.times.10.sup.6/kg CD34 cells,
0.55.times.10.sup.6/kg .alpha..beta.-TCR cells and
0.35.times.10.sup.6/kg hFC. He tolerated the conditioning very well
and engrafted and chimeric (88% donor cells) at one month based on
FISH. His donor chimerism was 28% as of day 697 (FIG. 8A). Donor T
cell chimerism was 34% at day 501. The patient has remained
asymptomatic since his transplant and is producing predominantly
normal RBC (FIG. 8B). The reticulocyte counts for patient SCD #4
has ranged between 0.5% and 1%, which is within normal ranges (FIG.
8C).
Example 4
Summary
[0085] This section described successful transplantation of two
heavily-transfused SCD patients using HLA-identical marrow from
sibling donors. Both patients were successfully transplanted using
reduced-intensity non-myeloablative conditioning and have remained
disease free for >2 years. At enrollment, they were
transfusion-dependent and at very high risk for painful crises and
other complications. Both patients have been successfully weaned
from immunosuppression.
Section D--Treatment of Sickle Cell Disease in Humans
[0086] Five individuals at high risk for morbidity and mortality
from their thalassemia were enrolled on the protocol according to
the inclusion and exclusion criteria below.
Example 1
Inclusion Criteria
[0087] The following criteria were established to identify
individuals with thalassemia who have a high predicted morbidity
and are at risk for early mortality: patients with alpha or beta
thalassemia major; or patients with other complex and
transfusion-dependent hemoglobinopathies. Individuals must also
meet all of the following general inclusion criteria: individuals
must have a related donor (identical or mismatched for 1, 2 or 3
HLA-A, -B or -DR loci); individuals must have adequate
cardiopulmonary function as documented by echocardiogram or
radionuclide scan (shortening fraction >26% or ejection fraction
>40% or >80% of normal value for age); individuals must have
adequate pulmonary function documented by FEV 1 of .gtoreq.50% of
predicted for age and/or DLCO (corrected for hemoglobin)
.gtoreq.50% of predicted for age for patients >10 years of age
(if patient cannot perform PFT's, resting pulse oximeter >85% on
room air or clearance by the pediatric or adult pulmonologist is
required); individuals must have adequate hepatic function as
demonstrated by a serum albumin >3.0 mg/dL, and SGPT or
SGOT<5 times the upper limit of normal; and individuals must
have adequate renal function as demonstrated by a serum creatinine
<2 mg/dL. If serum creatinine is >2 mg/dL, then a creatinine
clearance test or nuclear medicine GFR should document GFR of
.gtoreq.50 ml/min/1.73 m.sup.2. There are no age limits for this
protocol.
Example 2
Exclusion Criteria
[0088] Individuals are excluded from this trial if they meet any of
the following criteria: the individual lacks related donors; the
individual has uncontrolled infection or severe concomitant
diseases, and may not tolerate reduced intensity transplantation;
the individual exhibits severe impairment of functional performance
as evidenced by a Karnofsky (patients >16 years old) or Lansky
(children <16 years old) score of <70%; the individual
exhibits renal insufficiency (GFR <50 ml/min/1.73 m.sup.2); the
individual has a positive human immunodeficiency virus (HIV)
antibody test result; the individual is pregnant as indicated by a
positive serum HCG test; the individual's only donor is pregnant at
the time of intended transplant; the individual is of childbearing
potential and is not practicing adequate contraception; the
individual has been exposed to previous radiation therapy that
would preclude TBI; the individual is a Jehovah's witness; the
individual has uncontrolled hypersplenism; or the individual
exhibits severe alloimmunization with inability to guarantee a
supply of adequate PRBC donors.
Example 3
Recipient Evaluation
[0089] A complete history and physical examination of the
individual is performed. Estimation of pre-HSCT Lansky or Karnofsky
status is obtained. The history includes: age of diagnosis, overall
growth and development, frequency and number of transfusions, any
aplastic crises, prior treatment (e.g., hydroxyurea), baseline HbF
plus A2 levels, alloimmunization status, treatment and dates, any
MRI scans, transfusion therapy, infections, aseptic necrosis,
history of hepatitis, iron overload, prior liver biopsies, and
pathologic findings.
[0090] The following hematological tests are performed: CBC (Hgb,
Hct, MCV, MCHC, RDW, platelet, white blood cell count),
differential count, reticulocyte count, ferritin, folate,
quantitative Hgb electrophoresis, PT, PTT, fibrinogen, direct and
indirect Coombs test. In addition, the alpha gene number is
determined, the beta-globin haplotype is determined, globin chain
synthetic studies are performed, and the subject is ABO Rh typed
and screened.
[0091] The following chemistries are obtained: total and direct
bilirubin, SGPT,SGOT, alkaline phosphatase, Protein C, IgG
subclasses, albumin, Ca++/PO4++/Mg++, serum electrolytes,
BUN/creatinine, urinalysis, creatinine clearance/GFR; and endocrine
levels of T4, TSH, FSH, LH, and growth hormone.
[0092] The individual is HLA typed (HLA A, B, C, DQ and DR typing)
based on molecular analysis.
[0093] The following diagnostic tests are performed on the
individual: a CT scan (brain, sinuses, chest, abdomen, pelvis),
PFTs (crying vital capacity for younger children unable to perform
conventional PFT, DLCO for patients >10 years), EKG,
echocardiogram or MUGA scan, liver and spleen scan, ultrasound of
gall bladder, bone age, and estradiol or testosterone.
[0094] The individual is screened for the following infectious
markers: CMV, IgG, PCR, HSV & VZV IgGs, HIV 1 and 2 antibody
and PCR, HTLV 1 and 2 antibody, Hepatitis B surface antigen,
Hepatitis B core antibody, Hepatitis C antibody and PCR, EBV IgG
and IgM, toxoplasma IgG and IgM, West Nile Virus NAT, Trypanosoma
cruzi (Chagas) antibody, RPR or equivalent.
Example 4
Donor Evaluation and Selection
[0095] HLA-identical donor and recipients are used, or donor and
recipient mismatched pairs (e.g., up to haploidentical (parent,
aunt, uncle, cousin, or sibling)) are used. Family members willing
to donate bone marrow are HLA-typed. The best available match is
selected. All donors participating are evaluated as per FDA
regulations for donor screening prior to stem cell harvest. All
evaluations are completed within 30 days of the transplant.
Pediatric donors are considered for mobilization. If the donor is
not a good candidate for apheresis, bone marrow is harvested from
the iliac crest. If more than one related donor is available, the
closer matching, younger, and/or CMV-negative donor is selected.
All donors are placed on iron replacement therapy. Pheresed donors
can be supplemented with Vitamin K and/or calcium.
[0096] Donors are screened as described herein and the following
information is obtained. The history and physical examination of
the donor is obtained including pregnancy and transfusion history.
Donors are screened for CBC, differential; PT with INR, PTT and
fibrinogen; ABO and Rh Type and screen, ferritin, iron and TIBC;
HLA typing: HLA class I (-A, -B, -C) and class II (-DR, -DQ) typing
by molecular analysis; hemoglobin electrophoresis (thalassemia
trait is acceptable); SGPT or SGOT, alkaline phosphatase, and
bilirubin (total and direct); serum pregnancy test; serum
electrolytes, BUN, and creatinine; CMV, IgG, PCR, HSV & VZV
IgG, HIV 1 and 2 antibodies and PCR, HTLV 1 and 2 antibodies,
Hepatitis B surface antigen, Hepatitis B core antibody, Hepatitis C
antibody and PCR, EBV IgG and IgM, Toxoplasma IgG and IgM, West
Nile Virus NAT, Trypanosoma cruzi (Chagas), RPR or equivalent test;
hepatitis B core antibody (if antibody-positive, perform PCR for
viral DNA, accept donor if negative); hepatitis B surface antigen
(reject Hepatitis B antigen positive donor); HCV antibody (positive
donor is acceptable only if PCR for viral DNA is negative); Herpes
Simplex Virus antibody (document status only; positive donor is not
rejected); HIV I/II antibody (reject HIV I/II positive donor); HIV
PCR (reject HIV PCR positive donor); HIV I/II antibody (reject HTLV
I/II positive donor); CMV antibody titer (if positive and recipient
is negative, consider another donor if available, otherwise CMV
screening and prophylaxis is mandatory);
[0097] serologic test for syphilis (if positive, perform a
fluorescent treponemal antibody test; donor is accepted if
fluorescent treponemal antibody is negative); chest X-ray, if the
donor is greater than 21 years of age; and EKG if the donor is
greater than 40 years of age.
Example 5
Pre-Transplantation Treatment of Donor
[0098] For donors, a total of 560 cc of blood will be collected for
archiving of lymphocytes for immunocompetence testing. This can be
obtained as a single blood donation pre-transplant (450 cc). The
remaining eleven 10 cc-yellow top collection tubes are obtained
eight weeks after the first donation. For pediatric bone marrow
donors, no more than 3 ml/kg at any one time are drawn, and no more
than 7 ml/kg over a six-week period are drawn as per the NIH
guidelines for pediatric research blood draws.
[0099] Beginning day-4 (with respect to HSC+hFC infusion) and for
up to +4 days, 10 .mu.g/kg G-CSF is administered b.i.d. Collection
begins on day-1. A minimum of 5.times.10.sup.6 CD34/kg total is
collected. A maximum of two collections are done. With each blood
stem cell donation, 5-10 ml of blood is taken at the start and at
the end of the procedure to measure blood cell counts including
enumeration of CD 34+ cells.
[0100] At two days and one week after donation, the donor is
contacted to confirm whether any adverse events have occurred. The
donor also is asked to donate a blood sample (7 .mu.l) one month
after donation to ensure blood counts have recovered. The donor is
treated with therapeutic iron, Vitamin K or calcium as needed. The
visits for G-CSF administration, blood stem cell donations, and
blood draws are summarized below in Table 4 (e.g., an X marks what
will occur on each visit).
TABLE-US-00004 TABLE 4 Symptom Blood Stem Assess- Filgrastim/ Cell
Blood Visits ment G-CSF Donation Draws Screening X X Preparation,
Day -3 X X X Preparation, Day -2 X X Preparation, Day -1 X X
Preparation, Day 0, First X X X X donation Second donation* X X X 2
days after donation X 1 week after donation X Potential blood draws
to X test for donor chimerism in the recipient (up to 3 years) *2nd
donation occurs only if sufficient cells are not obtained in the
1st collection
Example 6
Recipient Conditioning
[0101] Individuals are examined by a radiation therapist to
determine dosimetry for TBI. Central venous access is established
in all patients prior to initiation of conditioning. Campath-1H is
administered in a first session at day-53, -52, -51, and -50 at a
maximum dose of 30 mg and in a second session at a maximum dose of
20 mg administered at day-24, -23, -22, and -21. The pediatric dose
of Campath is 10 mg/day on cycle one and 7 mg/day on cycle two. For
smaller recipients and those less than one year of age, Campath-1H
is dosed at a rounded up dose of 0.4 mg/kg for the first regimen,
and at a rounded up dose of 0.3 mg/kg for the second regimen. The
route of administration of the Campath, either subcutaneously or
intravenously, is at the discretion of the attending physician.
Start dates for Campath administration can be moved forward or
backward 1-3 days to accommodate scheduling conflicts. Fludarabine
is administered on day-5, -4, and -3. The individual receives TBI
and begins cyclosporine immunosuppression at day-1. The second
immunosuppressive medication, mycophenolate mofetil, is started the
evening of HSC+hFC infusion (day 0). The conditioning regimen is
shown in the following Table.
TABLE-US-00005 TABLE 5 Conditioning Approach Day -53 to Campath-1H
is administered at 30 mg/day for adults -50 and 10 mg/day for
children over each of the four days. Day -24 to Campath-1H is
administered at 20 mg/day for adults -21 and 7 mg/day for children
over each of the four days. Day -5 -4, Fludarabine is administered
at 30 mg/m2 intravenously -3 over a period of 30 minutes on each of
these three days. Day -1 Pre-transplant conditioning 200 cGy TBI
(35-40 cGy/ min); Cyclosporine is administered day -1 and continued
until it has been determined that the patient has engrafted, or it
has been demonstrated that the patient has failed to engraft, or at
the discretion of the physician. If engraftment occurs,
cyclosporine is continued for at least 12 months. If there is no
engraftment, cyclosporine is discontinued. Marrow is processed to
retain hFC and HSC using ferromagnetic approach. Day 0 HSC + hFC is
administered. MMF is started.
[0102] The radiation is delivered at day-1. The radiation dose is
200 cGy of 6 MV accelerator X-rays, delivered in one fraction. A
dose rate of 35-40 cGy/minute is used, dependent on the distance,
energy, and patient dimensions. Dose variations greater than 10%
are evaluated and approved on an individual basis. Infusion of the
HSCs+hFCs occurs on day 0. Patients receive daily penicillin or
equivalent prophylaxis for 2 years post-transplant, or longer at
the discretion of the treating physician.
Example 7
HSC+hFC Cell Processing
[0103] The mobilized peripheral blood stem cells are incubated with
monoclonal antibodies that are specific for alpha beta TCR T cells
and B cells, then depleted by immunomagnetic separation. The
composition of the infused cells is assessed by immunofluorescent
staining for CD34 HSCs; CD8+/TCR-/CD56.sup.dim/neg hFCs;
.gamma..delta.T cells, and .alpha..beta.-TCR+ T cells. The adequacy
of cellular depletion is determined by flow cytometric analysis,
and the clinician is notified of preliminary cell doses prior to
infusion. The cell product also is analyzed for bacteria, fungus,
and endotoxins. The HSC+hFC product is infused via a central venous
line in a monitored setting per institutional guidelines.
[0104] The processed graft is administered to all subjects, and the
graft is only limited based on the maximal allowable alpha beta TCR
dose. However, only those subjects with a minimally acceptable
graft (e.g., at least 5.times.10.sup.9 total leukocytes available
from the collection to process; at least 5.times.10.sup.6 CD34/kg
of recipient body weight; and a T cell depletion of less than 0.5
logs) are evaluated as described herein.
Example 8
Cell Dosing Algorithm
[0105] As many HSCs, hFCs and progenitors as possible are
administered within the context of a maximal allowable T cell dose
to avoid GVHD. Presently, the maximum dosing is 3.0.times.10.sup.6
to 4.2.times.10.sup.6 alpha beta T cells/kg recipient body weight
(with a preferred starting point at 3.8.times.10.sup.6 alpha beta T
cells/kg recipient body weight). Recipients are followed for a
minimum of 28 days. If engraftment is not observed, the maximal
allowable alpha beta TCR dose is increased by one unit
(4.times.10.sup.5/kg recipient body weight). The maximal allowable
alpha beta TCR dose is increased until stable engraftment is
achieved without significant GVHD. For HLA-matched transplants,
there is no maximum T cell cap and cell dose does not increase
based on the outcome of these matched transplants. For patients who
are mismatched, the maximal allowable alpha beta TCR dose is
determined.
TABLE-US-00006 TABLE 6 HSC, hFC, progenitors As many as possible NK
cells, B cells Record and report doses .gamma..delta.-TCR + T cells
Record and report doses .alpha..beta.-TCR + T cells For HLA
matched, there will be no cap. For HLA mismatched, the maximal
allowable will be determined by the last safe dose in the kidney,
heart, liver tolerance, sickle cell, and MS protocols.
[0106] If significant (>0.5%) donor engraftment is observed in
the first 28 days, the individual is followed for an additional 28
days to assess the incidence of acute GVHD.
Example 9
Additional Sickle Cell Patients Transplanted
[0107] Subject #5 was 9 years of age at the time of transplant
(March 2006). He had experienced multiple pain crises, two episodes
of acute chest syndrome before transplant, and had been treated
with exchange transfusions for 7 years. The subject received
HLA-matched trait sibling donor's iliac crest bone marrow and was
conditioned with essentially the same regimen as described in
Section C above. The graft contained 5.24.times.10.sup.6 CD34+
cells/kg of body weight, 0.55.times.10.sup.6 alpha
beta-TCR+cells/kg body weight and 0.35.times.10.sup.6 FC cells/kg
body weight. The subject has been transfusion-independent post-stem
cell transplant with 100% donor RBC production and chimerism levels
at 20-30% donor by FISH for greater than 1525 days post-transplant.
Immunosuppression was discontinued at 23 months post-transplant.
Subject has not exhibited graft-versus-host disease (GVHD),
transplant-related toxicity, or sickle cell complications since
transplant.
[0108] Subject #7 was a 16-year-old male who experienced repeated
acute chest syndrome episodes that required red blood cell
transfusion therapy. Prior to undergoing the transplant in
September 2009, he was hospitalized for osteomyelitis of the right
knee and multiple vaso-occlusive painful events. The subject
received a haploidentical transplant from his parent. The subject
was conditioned essentially as described above in Section C. The
subject tolerated the conditioning well and the transplant was
uneventful. He received 3.26.times.10.sup.6 CD34+ cells/kg body
weight, 3.8.times.10.sup.6 alpha beta TCR+ cells/kg body weight,
and 0.5.times.10.sup.6 FC cells/kg body weight, and he was managed
as an outpatient. Unfortunately, this subject was not compliant in
the immediate post-transplant period and did not regularly take
cyclosporine and MMF as required. Chimerism was not present at
post-transplant months 1 and 2. Post-transplant, he experienced a
recurrent pain crisis that subsequently resolved. The subject
remains on the study to monitor for adverse events, but chimerism
testing was discontinued after post-transplant month two.
[0109] Subject #8 was a 12-year-old female who experienced numerous
hospitalizations for pain crises. She also had undergone a
splenectomy following sequestration and cholcystectomy. She
underwent conditioning essentially as described above in Section C,
and she received a haploidentical transplant from her parent, who
had the SCD trait. She received 19.1.times.10.sup.6 CD34+ cells/kg
body weight, 3.8.times.10.sup.6 alpha beta TCR+ cells/kg body
weight, and 0.79.times.10.sup.6 FCs/kg body weight, and, following
transplantation, she was managed as an outpatient. She tolerated
the conditioning very well and demonstrated robust donor
engraftment of 71% at one month post-transplant. Her whole blood
chimerism remained durable at 84%, with lymphoid chimerism at 58%
and myeloid chimerism at 95% at month nine. She was producing 100%
donor RBC as reflected by hemoglobin A at 57%, hemoglobin S at 41%,
and hemoglobin A2 at 2% as demonstrated by hemoglobin
electrophoresis. The subject has not required transfusion therapy
since transplant and is asymptomatic. She has had no evidence for
GVHD.
[0110] Subject #9 was a 25-year-old male who experienced repeated
PRBC transfusions, cholecystectomy with sickle cell disease,
hypertension and renal vascular disease prior to transplant. The
subject was conditioned essentially as described above in Section
C, and he tolerated the conditioning well. The alpha beta TCR+
cells for this subject was increased to 4.2.times.10.sup.6 cells/kg
body weight, and the subject also received 1.46.times.10.sup.6
CD34+ cells/kg body weight and 0.72.times.10.sup.6 FCs/kg body
weight. He demonstrated 10% donor chimerism at post-transplant
month one. His chimerism decreased to 4% at month two and to less
than 2% at day 100. The subject was admitted for elevated
creatinine due to calcineurin inhibitor (CNI) sensitivity in the
second post-transplant month. The dose was adjusted and the SAE
resolved. About one month later, he was admitted for fever, gram
positive cocci, and CMV infection. He went off study to participate
in an investigational drug for CMV treatment.
Section E--Prevention of Graft vs. Host Disease (GVHD) Following
Solid Organ Transplant
Example 1
Patient Recruitment
[0111] Candidates for the protocol were selected from the list of
patients awaiting renal transplantation or who were being evaluated
for transplantation. This selection process was carried out by the
transplant surgeons and the transplant nurse coordinators of the
Institute of Cellular Therapeutics at the University of Louisville
("the Institute").
Example 2
Inclusion Criteria
[0112] A candidate patient must be between the ages of 18 and 65
years and meet the Institution's criteria for renal transplantation
for end-organ failure. A candidate patient must be receiving his or
her first renal transplant. A candidate patient must be receiving a
renal transplant only. The crossmatch must be negative between the
donor and the recipient. Women who are of child bearing potential
must have a negative pregnancy test (urine test is acceptable)
within 48 hours prior to initiating TBI and must agree to use
reliable contraception for 1 year following transplant. Candidate
patients must exhibit no evidence of donor-specific antibody,
presently or historically.
Example 3
Exclusion Criteria
[0113] Patients are not candidates if they have a clinically active
bacterial, fungal, viral or parasitic infection, or if they are
pregnant. Patients are not eligible if they exhibit clinical or
serologic evidence of viral infection that would preclude the
recipient from receiving a kidney transplant. A patient is not a
candidate if they have received previous radiation therapy at a
dose which would preclude TBI, if there is a positive crossmatch
between the donor and the recipient, or if there is evidence for
immunologic memory against the donor. Patients also are excluded if
their body mass index (BMI) is less than 18 or greater than 35.
Example 4
Donor Selection Criteria
[0114] Donors for this protocol must meet all of the Institute's
criteria for renal and stem cell transplant.
Example 5
Protocol
[0115] The timing for all manipulations is relative to the TBI
conditioning of the recipient on day 0. Beginning day-3 and for up
to four days, 10 .mu.g/kg G-CSF was administered b.i.d. Collection
began on day 0. On day 0, a CD34 count was performed prior to
giving the final dose of G-CSF. HSC+hFC transplantation was
scheduled 4 to 6 weeks prior to the desired date of kidney harvest.
The donation and transplant of the kidney is not schedules until
the donor's platelet counts have returned to baseline and safe
levels for kidney donation (e.g., greater than 100,000/.mu.l of
whole blood).
[0116] The visits for G-CSF administration, blood stem cell
donations, and blood draws are summarized in Table 7 The `X` marks
what will occur on each visit.
TABLE-US-00007 TABLE 7 Donor Mobilization Symptom Blood Stem
Assess- Filgrastim/ Cell Blood Visits ment G-CSF Donation Draws
Screening X X Preparation, Day -3 X X X Preparation, Day -2 X X
Preparation, Day -1 X X Preparation, Day 0, X X X X First donation
2 Days after donation X 1 Week after donation X Potential blood
draws to X test for donor chimerism in the recipient (up to three
years)
Example 6
Pre-Transplant Conditioning
[0117] Cell dose (HSC+hFC) as well as degree and type of
conditioning of the recipient were independent variables that
influence engraftment. In the current protocol, cell dose and
conditioning were optimized until >1% donor chimerism was
established. The initial target cell dose for HSC+hFC was
.gtoreq.1.times.10.sup.8 CD34+/kg. The first patients received 200
cGy TBI, fludarabine (30 mg/m.sup.2 day-3 to -1), and
post-transplant immunosuppression with MMF (15 mg/kg q 12 h
beginning day 0), and FK506 (0.02 mg/kg q 12 h beginning day -1)
for six months, or as clinical need required. The decision to use
either FK506 (tacrolimus) or cyclosporine was left to the physician
because patients differ in their ability to tolerate either drug.
The marrow was infused on day+1. The schedule is shown in Table
8.
TABLE-US-00008 TABLE 8 Day Treatment Dose -3 Fludarabine after
dialysis (if required) 30 mg/m2 -2 Fludarabine after dialysis (if
required) 30 mg/m2 -1 Fludarabine 30 mg/m2 0 Start MMF and FK506 or
cyclosporine 0 TBI (200 cGy) 35-40 cGy/min Harvest donor marrow and
process to obtain HSC + hFC +1 Infuse HSC + hFC after dialysis (if
required) +28- Renal transplantation; continue MMF and calcineurin
60 inhibitors
[0118] If the recipient required dialysis, the dosing of
fludarabine and the HSC+hFC infusion occurred after dialysis on the
specified days. On the morning of HSC+hFC infusion, an extra liter
of volume was removed from dialysis to account for the volume of
HSC+hFC. Dialysis then was scheduled for 48 hrs or later following
HSC+hFC infusion to give the cells an optimum opportunity to home
to the marrow compartment.
Example 7
Outcomes
[0119] A minimum of about 2 weeks post-HSC+hFC infusion or as long
as the recipient needs to fully recover from the stem cell
transplant procedure passed before the renal transplant was
performed from the same donor. The following algorithm was used
based on the outcome of the HSC+hFC transplant.
[0120] 1) if the recipient exhibited chimerism of .gtoreq.1% and
was determined to be tolerant to the donor, at least six months of
Prograf and MMF was administered while donor:host tolerance to the
kidney is established. The recipient did not receive Campath-1H or
any additional immunosuppression at the time of transplant.
[0121] 2) if the recipient did not engraft, the patient as tested
by flow crossmatch prior to transplant to ensure no donor-specific
antibodies developed. If no donor-specific antibody was present,
the patient underwent living donor kidney transplant using
conventional lymphodepletion induction with Campath-1H followed by
maintenance immunosuppression with FK506 and MMF. Other
lymphodepletion approaches for induction therapy such as ALG can be
used in place of Campath per standard of care.
[0122] 3) if donor-specific antibody developed, the patient was
assessed and a clinically appropriate antibody reduction protocol
implemented prior to transplantation. Patient sensitization was not
expected.
Example 8
Cell Dosing Algorithm
[0123] The goal of the study was to engineer a graft with adequate
HSCs, hFCs and progenitors for allogeneic engraftment while
avoiding GVHD. A cell dosing algorithm was established that is tied
to the maximum allowable alpha beta T cell dose. For example, if
toxicity (GVHD) did not occur but engraftment was not durable, the
maximum allowable T cell dose was increased by 1 unit (see below).
The maximal allowable T cell dose containing as many HSCs, hFCs,
and progenitors was administered. The algorithm that was used is
shown in Table 9.
TABLE-US-00009 TABLE 9 HSC, hFC, As many as possible progenitors
alpha beta For HLA matched, there is no cap. For HLA TCR + T
mismatched, the maximal allowable is cells determined by the last
safe dose in the kidney, heart, liver tolerance, sickle cell, and
MS protocol NK cells, B Record and report doses cells gamma delta
Record and report doses TCR + T cells
[0124] The present cell dosing currently allows a maximum of
3.0.times.10.sup.6 to 4.2.times.10.sup.6 alpha beta T cells/kg
recipient body weight; 3.8.times.10.sup.6 alpha beta T cells/kg
recipient body weight was the starting dose. Each patient was
followed for at least 28 days. If no evidence of engraftment was
observed, the maximal allowable alpha beta-TCR dose was increased
by one unit (4.times.10.sup.5/kg recipient body weight). The
maximal allowable alpha beta-TCR dose was increased in subjects
until stable engraftment was achieved without significant GVHD. For
HLA-matched transplants, there was no maximum T cell cap and cell
dose was not increased based on the outcome of the matched
transplants. For patients who are mismatched, the maximal allowable
alpha beta-TCR dose was determined.
[0125] If significant (>0.5%) donor engraftment was observed in
the first 28 days, the subject was followed for an additional 28
days to assess the incidence of acute GVHD before the cell dose was
increased. It was expected that the majority of cases of acute GVHD
would be apparent by 8 weeks post-transplant. If evidence of severe
GVHD was seen, the maximal allowable T cell dose was reduced to a
level proven safe. To date, GVHD has not been observed.
Example 9
HSCs+hFCs
[0126] Donor PBMC was processed to enrich for hFCs and HSC.
Approximately 85% of total bone marrow composition was removed,
including GVHD-producing T-cells and B-cells, using a ferromagnetic
approach. The resultant product was enriched for hFCs, HSCs and
progenitors. After the adequacy of the processing was confirmed by
flow cytometry, the HSC+hFC graft was approved for infusion. A
delay in the bone marrow transplantation of up to 72 hours
following donor cell harvest was accepted to allow for bone marrow
processing and transplantation. Dose-adjusted Bactrim and Valcyte
(if CMV+) or Valtrex (if CMV-) prophylaxis was started. The patient
was carefully monitored during and after infusion of marrow to
detect any changes in, for example, respiration, blood pressure, or
angioedema, which may be an indication of hypersensitivity.
Example 10
Post-Transplant Immunosuppression
[0127] Subjects enrolled in this protocol received standard
immunosuppression at the discretion of the attending physician and
according to institutional protocol. For deceased donor
kidney/HSC+hFC recipients and living donor HSC+hFC recipients
without demonstratable donor chimerism at 1 month, this generally
included Prograf plus MMF after lymphodepletion induction therapy
with ALG or Campath. Prograf levels were maintained between 8-12
ng/ml. Starting on day 0, MMF was generally dosed at 1-1.5 gms
b.i.d. A one time dose of Campath, 30 mg IV, was optionally given
in the operating room. SoluMedrol was given at a dose of 500 mg IV
in the operating room one hour prior to the Campath dose, then
post-operatively on day 1 at a dose of 250 mg IV and on post op day
2 at a dose of 125 mg. FK506 and MMF were continued for at least 6
months in patients who were chimeric to promote engraftment and
tolerance induction.
Example 11
Preliminary Solid Organ Transplant Protocol
[0128] Preliminary experiments demonstrated the success and safety
of HSCs+hFCs and immune-based nonmyeloablative conditioning in
renal transplant recipients. Dosing of the HSCs+hFCs was performed
to maximize safety and optimize HSC and hFC content. The overall
goal was to completely avoid GVHD. Solid organ tolerance protocols
were begun with a maximum of 0.2.times.10.sup.6 T cells/kg
recipient body weight to perform a dose-escalation. Total alpha
beta T cells were used in the dose-escalation experiments since the
other effector cells for GVHD (NK, B-cells, and APC) are all
present in amounts proportional to total alpha beta T cells. CD34
and hFC dose were optimized within the maximum allowable T cell
dose. No significant immunologic events (i.e., rejection episodes
or antibody production) were observed in any of the patients. None
of the patients developed GVHD, but only transient chimerism was
observed.
[0129] Since September 2004, nine heart and eleven kidney patients
have been transplanted using the dose-escalation strategy set forth
in Table 10. Patients 5, 6, 12, and 13, which are highlighted in
Table 10, are described in more detail; three patients underwent
simultaneous kidney/HSC+hFC transplantation from living donors and
the remaining patient received his kidney from a deceased donor.
All four patients were conditioned with 200 cGy TBI and underwent
lymphodepletion induction therapy with Campath followed by
maintenance immunosuppression with MMF and a calcineurin inhibitor.
They did not receive fludarabine. The 4 patients are briefly
described below.
[0130] Patient #5 is a 55-year-old male who underwent a deceased
donor renal allograft/HSC+hFC transplant in Sept 2005. The patient
received 3.7.times.10.sup.6 CD34 and 0.8.times.10.sup.6 hFC per kg
recipient body weight. The conditioning was well tolerated and no
adverse events related to the approach occurred. The donor and
recipient shared a 1/6 HLA-antigen match. The patient experienced
the anticipated nadir, and then recovered immune function and
endogenous hematopoiesis. He is doing well with a recent serum
creatinine of 2.1.
[0131] Patient #6 is a 58-year-old male who underwent a living
donor kidney/HSC+hFC transplant from an unrelated friend in
November 2005. The patient received 1.33.times.10.sup.6 CD34 and
0.18.times.10.sup.6 hFC per kg recipient body weight. The
conditioning was well tolerated and no adverse events related to
the approach occurred. The donor and recipient shared a 1/6
HLA-antigen match. The patient experienced the anticipated nadir,
and then recovered immune function and endogenous hematopoiesis. He
is doing well, with a recent serum creatinine of 2.0.
[0132] Patient #12 is a 37-year-old female who underwent a living
donor kidney/HSC+hFC transplant from her cousin in October 2007.
She received 2.24.times.10.sup.6 CD34 and 0.41.times.10.sup.6 hFC
per kg recipient body weight. The conditioning was well tolerated
and no adverse events related to the approach occurred. The donor
and recipient shared a 2/6 HLA-antigen match. The patient
experienced the anticipated nadir, and then recovered immune
function and endogenous hematopoiesis. She is doing well with a
recent creatinine of 1.2.
[0133] Patient #13 is a 49-year-old female who underwent a
kidney/HSC+hFC transplant from her brother in November 2007. The
patient received 3.85.times.10.sup.6 CD34 and 0.78 10.sup.6 hFC per
kg recipient body weight. The conditioning was well tolerated and
no adverse events related to the approach occurred. The donor and
recipient shared a 4/6 HLA-antigen match. The patient experienced
the anticipated nadir, and then recovered immune function and
endogenous hematopoiesis. She is doing well with a recent
creatinine of 1.2.
TABLE-US-00010 TABLE 10 Cell dosing for patients (per kg recipient
weight) Trans- Date of Trans- TBI T # plant plant Source (cGy)
cells* CD34* hFC* 1 Heart September 2004 VB 200 0.2 1.99 0.23 2
Kidney October 2004 MPB 200 0.2 0.78 0.02 3 Heart December 2004 VB
200 0.4 5.70 1.07 4 Kidney February 2005 VB 200 0.6 3.80 0.14 5
Kidney September 2005 VB 200 0.8 3.70 0.80 6 Kidney November 2005
MPB 200 1.0 1.33 0.18 7 Heart March 2006 VB 200 1.2 0.71 0.08 8
Heart March 2006 VB 200 1.2 5.60 0.74 9 Heart May 2006 VB 200 1.4
4.69 0.55 10 Heart February 2007 VB 200 1.8 3.81 0.72 11 Heart
August 2007 VB 200 1.8 2.08 0.75 12 Kidney October 2007 MPB 200 2.2
2.24 0.41 13 Kidney November 2007 MPB 200 2.2 3.85 0.78 14 Heart
January 2008 VB 200 2.6 2.67 1.61 15 Kidney March 2008 MPB 200 3.0
9.26 9.34 16 Kidney April 2008 MPB 200 3.0 1.45 5.81 17 Heart April
2008 VB 200 3.4 4.86 1.87 18 Kidney February 2009 IC 200 0.96 0.90
0.16 19 Kidney April 2009 MPB 200 3.8 2.53 4.48 20 Kidney May 2009
MPB 200 3.8 3.60 0.90 *.times.10.sup.6 cells; VB = vertebral body;
MPB = mobilized peripheral blood; IC = iliac crest
Example 12
Results of Preliminary Protocol
[0134] Initially, the chimerism observed was low (<0.2%) and
only transient.
[0135] However, as the total cell dose was increased, durable mixed
chimerism was achieved. The immune response to the graft was
modulated by the marrow infusion as evidenced by transient
donor-specific tolerance in mixed lymphocyte reaction (MLR) assays
observed in the more recently transplanted patients and the absence
of any clinical or histologic rejection episodes.
[0136] The fact that endogenous hematopoiesis resumed in those
patients who did not engraft confirmed the non-myeloablative nature
of the conditioning. There was an expected nadir of absolute
neutrophil count (ANC) of less than 1,000 that occurred in the
recipients between 7 and 18 days (FIGS. 9A and 11A), which was
managed as an outpatient in Patient #12. It was found that
administration of G-CSF did not accelerate recovery. The MMF and
FK506 was continued through the nadir to promote engraftment.
Recovery of B cells (CD19), CD4+ cells, and CD8+ cells occurred by
3 months in the Campath-lymphodepleted recipient #12 (FIG. 11B).
Platelet counts were determined following solid organ transplant
(FIGS. 9B, 10A, and 11C). The platelet nadir, if present, typically
was brief and usually did not require transfusion therapy. The
chimerism that was established in solid transplant patients is
shown in FIGS. 9C and 10B.
Example 13
Modified Protocol for Solid Organ Transplant
[0137] The kidney/HSC +hFC transplant protocol was modified to add
fludarabine conditioning and to perform the living donor
transplants sequentially, with HSC +hFC administered one month
prior to the kidney graft placement.
[0138] The first transplant (stage 1 FCRx) was performed in March
2008 (Patient #15 in Table 10). She is a 31-year-old female whose
husband was her donor. The patient is currently in her nadir period
and is doing well as an outpatient. A flow crossmatch performed on
day 14 was negative (Table 11). In this assay, the binding of
antibodies to donor T and B cells was measured by flow cytometric
analysis in MCDF units.
TABLE-US-00011 TABLE 11 Flow Crossmatch T cell B cell % T Positive/
% B Positive/ cells MCDF Negative cells MCDF Negative Negative 57
250 - 5 301 - control Positive 52 495 + 5 534 + control Patient 54
245 - 5 246 - #15 Patient 52 252 - 5 266 - #15
[0139] These results demonstrated the safety of the
non-myeloablative conditioning and the feasibility of the HSC+hFC
process and product in solid organ transplant. It is noted that
none of the recipients became sensitized to the donor.
Section F--Kidney Transplant
Example 1
Donor and Recipient Eligibility
[0140] All protocols were approved by the Northwestern
Institutional Review Board, the FDA IND 13881, and informed consent
was obtained for all donors and recipients. Donors and recipients
had to meet Institutional criteria as suitable living transplant
donors and recipients; participants had to complete all phases of
the pre-transplant donor and recipient evaluation to be considered
for study participation. Inclusion criteria for transplant
recipients included age between 18 and 65 years, absence of any
donor-specific antibodies as assessed by flow PRA analysis, and
receiving only a living donor kidney transplant. Women of
childbearing age had to have a negative pregnancy test (urine
testing acceptable) within 48 hours of receiving TBI and agree to
use reliable contraception for a year after the transplantation.
Exclusion criteria included clinically active bacterial, fungal,
viral or parasitic infection, pregnancy, previous radiation therapy
at a dose which would preclude TBI, a positive flow cytometry
crossmatch between donor and recipient, presence of donor-specific
antibodies, body mass index (BMI)>35 or <18, and positive
serologies for HBV, HCV, and HIV.
Example 2
Conditioning and Donor Product Preparation
[0141] Conditioning consisted of three doses of fludarabine
(30/mg/kg/dose) at days -4, -3, -2; two doses of cyclophosphamide
(Cytoxan; 50 mg/kg/dose) at days-3 and +3; and 200 cGy TBI at day-1
relative to the renal transplant as depicted in FIG. 12.
Hemodialysis was performed 6-8 h after the administration of
fludarabine and Cytoxan. Tacrolimus (target trough concentrations
8-12 ng/ml) and mycophenolate mofetil (MMF) (Cellcept; 1 gm orally
twice daily if recipient weighs <80 kg, 1.25 gm twice daily if
recipient weighs >80 kg) were started at day-3 and continued
throughout. HSCs+hFCs can be administered to the recipient at day 0
(i.e., the same day as the transplant) or at day+1.
Example 3
Hematopoietic Stem Cell Collection
[0142] At least two weeks prior to the renal transplant, donors
were mobilized with granulocyte colony stimulating factor (G-CSF)
at 10 mcg/kg b.i.d. and apheresis was performed on day+4. The
product was transported by courier to the Institute for Cellular
Therapeutics (ICT) and processed to remove mature graft-versus-host
disease (GVHD)-producing cells while retaining hematopoietic stem
cells (HSC), facilitating cells (FCs), and progenitor cells. The
product was then shipped back to Northwestern University for
infusion, either as a fresh product or cryopreserved.
Example 4
Immunologic Monitoring
[0143] The recipient response to PHA, Candida, tetanus toxoid,
donor and third-party alloantigens was tested monthly (see, for
example, Patel et al., 2008, J Allergy Clin. Immunol.,
122:1185-93). Flow crossmatch assay to detect donor antibodies were
performed at 1 and 6 months. Chimerism testing was performed by
molecular assay using short tandem repeats (Akpinar et al., 2005,
Transplant., 79:236-9). Surveillance biopsies were performed at 1
year. At selected time points, imunophenotypic analysis of
peripheral blood was performed for T cell, B cell, NK cell,
monocytes, CD4+/CD25+ Fox P3+ regulatory T cell (T.sub.reg), and T
effector cell (T.sub.eff) recovery.
Example 5
Chimerism Testing
[0144] Chimerism was determined by genotyping of simple
sequence-length polymorphisms encoding short tandem repeats (STR).
For lineage chimerism testing, CD 19.sup.+ (B cells), CD3.sup.+ (T
cells), and CD66B.sup.+ myeloid cells were sorted from whole blood
then analyzed by molecular STR typing.
Example 6
Weaning of Immunosuppression
[0145] Prograf and MMF were continued per standard of care until 6
months post-transplant. At that point, if chimerism or
donor-specific tolerance were present, the MMF was first
discontinued, then the Prograf was tapered off over to
sub-therapeutic amounts the next few months (e.g., .ltoreq.3.0
ng/ml by 9 months). Prograf was discontinued at 12 months if
evidence of chimerism and/or in vitro donor specific
hyporesponsiveness is present.
Example 7
Results
[0146] A summary of Subject #1--Subject #9 is shown below in Table
12, and Table 13 shows the cell dosing regimens. A few of the
subjects are discussed in more detail as follows.
[0147] Subject #3 is a 43-year-old white male who developed ESRD
due to polycystic kidney disease. A 1-of-6 HLA matched unrelated
altruist was his donor. A total of 3.8.times.10.sup.6 alpha beta
TCR+ T cells, 2.53.times.10.sup.6 CD34 cells, and
4.48.times.10.sup.6 FC/kg recipient body weight cryopreserved
product were infused. The recipient demonstrated 95% donor
chimerism at 1 month, and chimerism fluctuated between 63% and 100%
over 18-months post-transplant (FIG. 13A). At 12 months,
multilineage testing revealed 100% B cell, T cell, and myeloid
production (FIG. 13B). Flow crossmatch was negative at 1 month and
6 months. At month 5, the recipient exhibited donor-specific
tolerance and immunocompetence to respond to third-party
alloantigen (FIG. 13C). This has persisted through 12 months. His
renal function has remained stable based on creatinine output (FIG.
13D). The subject exhibited a transient nadir between 6-15 days
(FIGS. 13E and 13F), which was managed as an outpatient.
[0148] Subject #5 is a 40-year-old male whose renal failure was
secondary to chronic glomerulonephritis. He underwent a combined
FC/renal transplant from a 1-of-6 HLA matched unrelated donor. His
product was comprised of 3.8.times.10.sup.6 alpha beta-TCR.sup.+ T
cells, 0.7.times.10.sup.6 FC, and 3.94.times.10.sup.6 CD34 cells/kg
recipient body weight. The nadir followed a pattern similar to the
prior subject. Chimerism was 100% at 1 month, 92% at 3 months, and
94% at month 5. A donor-specific tolerant profile began to emerge
at month 3, with responses to PHA and third-party alloantigen but
not to donor.
[0149] Subject #6 is a 39-year-old female who developed ESRD
secondary to reflux. She underwent a second renal transplant from a
2-of-6 HLA matched unrelated donor. The product consisted of
3.8.times.10.sup.6 alpha beta TCR.sup.+ T cells,
8.59.times.10.sup.6 CD34.sup.+, and 3.11.times.10.sup.6 FC cells/kg
recipient body weight. The recipient exhibited 100% donor chimerism
at 1 month.
TABLE-US-00012 TABLE 12 Summary of Kidney + hFC Patients HLA Date
of Subject Sex Age Match Transplant Original Disease Adverse Events
1 M 50 5/6 February 2009 Membranous recurrent disease at 1- yr
post-transplant; successfully treated with rituximab 2 M 56 3/6
April 2009 Hypertension febrile septic episode at 3-months post-
transplant, marrow failure, autologous HSCT rescue, sepsis and
allograft failure; now successfully re- transplanted with living
donor kidney 3 M 43 1/6 May 2009 PCKD drug rash, shingles 4 M 29
3/6 June 2009 Alports wound infection, sub- Syndrome clinical
rejection at one-year post- transplant 5 M 40 1/6 February 2010
Chronic GN flank cellulitis, wound seroma 6 F 39 2/6 March 2010
Reflux: 2.sup.nd none Transplant 7 M 35 3/6 April 2010 Hypertension
none 8 F 46 1/6 July 2010 PCKD i.v. site cellulitis 9 M 28 0/6*
September 2010 IgA Nephropathy hemolytic uremic syndrome due to
FK506, converted to sirolimus and resolved *1 minor antigen
match
TABLE-US-00013 TABLE 13 Cell Dosing for Patients % Composition
delivered chimerism Anti-donor (10.sup.6/kg recipient weight) at 1
Antibody alpha beta Patient Source* month Production T cells CD34
FC 1 IC* 30 No 0.963 .896 0.157 2 MPB* 95 No 3.8 2.53 4.48 3 MPB
100 No 3.8 3.6 0.90 4 MPB 25 No 1.94 1.00 0.49 5 MPB 100 No 3.8
3.94 0.716 6 MPB 100 No 3.8 8.59 3.11 7 MPB 100 No 3.8 16.9 1.16 8
MPB 100 NA 3.8 12.6 2.74 9 MPB 0 NA 3.8 5.07 2.12 *Source: IC,
iliac crest marrow; MPB, mobilized peripheral blood
Example 8
Summary of hFC and Living Donor Kidney Transplant
[0150] Of the 9 subjects transplanted, the non-myeloablative
conditioning was well-tolerated. In addition, the post-transplant
nadir period for all subjects was easily managed as an
outpatient.
[0151] Eight of the nine subjects demonstrated macrochimerism
following transplantation, ranging from 6% to 100% at 1-month.
Durable chimerism was achieved in the majority of subjects.
[0152] One subject has been weaned entirely off of
immunosuppression. Several subjects have exhibited evidence of
donor-specific hyporesponsiveness and are poised to be weaned from
immunosuppression. Subjects were immunocompetent to respond to
mitogen (PHA), Candida, and MHC-disparate third party
alloantigen.
[0153] None of the subjects developed GVHD despite the HLA
mistatching.
Section G. Metabolic Disorders
Example 1
Treatment of Inherited Metabolic Disorders
[0154] Subject #1 was a seven-year-old child with metachromatic
leukodystrophy. He received a 3 out of 6 HLA-matched transplant
from his father, who carries the trait for metachromatic
leukodystrophy. The subject was conditioned essentially as
described above in Section C, Example 2. He tolerated the
conditioning and infusion very well as an outpatient. He received
14.4.times.10.sup.6 CD34+ cells/kg body weight, 3.8.times.10.sup.6
alpha beta TCR+ cells/kg body weight, and 4.1.times.10.sup.6 FCs/kg
body weight. His nadir was brief and he did not require transfusion
therapy. His chimerism, by molecular STR, has ranged between
80%-98%. At 14 months post-transplant, the recipient exhibited no
GVHD. The MLR results for this subject demonstrated tolerance to
the donor and confirmed the likelihood of durable long term
engraftment. Pre-transplant, the subject's Arylsulfatase A enzyme
level was 3, compared to the donor's level of 50
post-transplantation. The subject's level was approximately 50 at
three and six months post-transplant, and was 88.6 at one year
post-transplant. This represents the enzyme level of a
phenotypically normal patient.
Other Embodiments
[0155] It is to be understood that while the methods and
compositions has been described in conjunction with the detailed
description thereof, the foregoing description is intended to
illustrate and not limit the scope of the invention, which is
defined by the scope of the appended claims. Other aspects,
advantages, and modifications are within the scope of the following
claims.
* * * * *