U.S. patent application number 15/347500 was filed with the patent office on 2017-03-02 for augmentation of cell therapy efficacy including treatment with alpha 1,3 fucosyltransferase.
The applicant listed for this patent is Targazyme, Inc.. Invention is credited to Lynnet Koh, Leonard Miller, Stephen D. Wolpe.
Application Number | 20170058261 15/347500 |
Document ID | / |
Family ID | 50881170 |
Filed Date | 2017-03-02 |
United States Patent
Application |
20170058261 |
Kind Code |
A1 |
Wolpe; Stephen D. ; et
al. |
March 2, 2017 |
Augmentation of Cell Therapy Efficacy Including Treatment With
Alpha 1,3 Fucosyltransferase
Abstract
Disclosed are methods, compositions of matter, and kits useful
for augmentation of cells through modification of cellular membrane
properties following ex vivo treatment.
Inventors: |
Wolpe; Stephen D.; (Boyds,
MD) ; Miller; Leonard; (Carlsbad, CA) ; Koh;
Lynnet; (Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Targazyme, Inc. |
Carlsbad |
CA |
US |
|
|
Family ID: |
50881170 |
Appl. No.: |
15/347500 |
Filed: |
November 9, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14182141 |
Feb 17, 2014 |
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15347500 |
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12997230 |
Dec 9, 2010 |
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PCT/US2009/046800 |
Jun 9, 2009 |
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14182141 |
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61060084 |
Jun 9, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/17 20130101;
C12N 2501/724 20130101; C12N 5/0638 20130101; C12N 5/0623 20130101;
C12N 5/0637 20130101; C12N 5/0647 20130101; A61K 2035/124 20130101;
C12N 5/0663 20130101; C12N 5/0006 20130101; C12Y 204/01065
20130101 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783; A61K 35/17 20060101 A61K035/17; C12N 5/00 20060101
C12N005/00 |
Claims
1. A method of enhancing homing and engraftment of one or more T
cells, the method comprising the step of: contacting a T cell
population with .alpha.1,3-fucosyltransferase VI ex vivo to
fucosylate at least one surface molecule on the T cell(s) to
enhance selectin mediated binding thereof.
2. The method of claim 1, wherein the T cell population is further
defined as an ex vivo expanded T cell population.
3. The method of claim 1, wherein the T cell population is further
defined as a heterogeneous population of T cells.
4. The method of claim 1, wherein the T cell population comprises
Regulatory T cells.
5. The method of claim 1, further comprising the steps of:
contacting the T cell population with a fucose carrier; and
combining said fucosylated T cells with a
pharmaceutically-acceptable carrier to provide a composition
capable of administration via a route selected from a group
comprising intravenously, intraarterially, intramuscularly,
subcutaneously, transdermally, intratracheally, intraperitoneally,
intravitreally, and combinations thereof.
6. The method of claim 5, wherein the fucose carrier is mixed with
alpha 1,3-fucosyltransferase VI prior to contacting said mixture
with the T cell population, and wherein said fucose carrier is
guanosine diphosphate fucose.
7. A method of enhancing homing and engraftment of one or more T
cells, the method comprising the step of: contacting a T cell
population with .alpha.1,3-fucosyltransferase VII ex vivo to
fucosylate at least one surface molecule on the T cell(s) to
enhance selectin mediated binding thereof.
8. The method of claim 7, wherein the T cell population is further
defined as an ex vivo expanded T cell population.
9. The method of claim 7, wherein the T cell population is further
defined as a heterogeneous population of T cells.
10. The method of claim 7, wherein the T cell population comprises
Cytotoxic T cells.
11. The method of claim 7, further comprising the steps of:
contacting the T cell population with a fucose carrier; and
combining said fucosylated T cells with a
pharmaceutically-acceptable carrier to provide a composition
capable of administration via a route selected from a group
comprising intravenously, intraarterially, intramuscularly,
subcutaneously, transdermally, intratracheally, intraperitoneally,
intravitreally, and combinations thereof.
12. The method of claim 11, wherein the fucose carrier is mixed
with alpha 1,3-fucosyltransferase VI prior to contacting said
mixture with the T cell population, and wherein said fucose carrier
is guanosine diphosphate fucose.
13. A method of enhancing homing and engraftment of a T cell
population, the method comprising the step of: administering an ex
vivo expanded population of fucosylated T cells to a patient in
need thereof, the population of cells being fucosylated by contact
with .alpha.1,3-fucosyltransferase VI and/or
.alpha.1,3-fucosyltransferase VII that fucosylated at least one
surface molecule on the T cells to enhance selectin mediated
binding thereof.
14. The method of claim 13, wherein the population of fucosylated T
cells is further defined as a heterogeneous population of
fucosylated T cells.
15. The method of claim 13, wherein the population of fucosylated T
cells comprises Regulatory T cells that have been fucosylated by
contact with .alpha.1,3-fucosyltransferase VI.
16. The method of claim 13, wherein the population of fucosylated T
cells comprises Cytotoxic T cells that have been fucosylated by
contact with .alpha.1,3-fucosyltransferase VII.
17. The method of claim 13, wherein the patient suffers from at
least one condition selected from the group comprising a
myelodysplastic syndrome, a stem cell disorder, a
myeloproliferative disorder, a lymphoproliferative disorder, a
phagocyte disorder, a histiocytic disorder, a liposomal storage
disease, a congenital immune system disorder, an inherited
erythrocyte abnormality, an inherited platelet abnormality, a
plasma cell disorder, a tumor, an autoimmune disease, and
combinations thereof.
18. The method of claim 13, wherein the patient in need of
treatment with the modified cell population suffers from at least
one condition selected from the group comprising peripheral
arterial diseases, ischemic limb injury, diabetes, heart disease,
liver disease, bone disease, muscular dystrophy, Alzheimer's
disease, ALS, multiple sclerosis, Parkinson's disease, spinal cord
injury, stroke, head trauma, infertility, and combinations
thereof.
19. The method of claim 13, wherein the population of fucosylated T
cells is administered via a route selected from a group comprising
intravenously, intraarterially, intramuscularly, subcutaneously,
transdermally, intratracheally, intraperitoneally, intravitreally,
and combinations thereof.
20. The method of claim 13, wherein the population of fucosylated T
cells is administered to a site of injury or proximal thereto.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS/INCORPORATION BY REFERENCE
STATEMENT
[0001] This application is a continuation of U.S. Ser. No.
14/182,141, filed Feb. 17, 2014; which is a continuation-in-part of
U.S. Ser. No. 12/997,230, filed Jun. 9, 2009, now abandoned; which
is a US national stage application filed under 35 USC .sctn.371 of
International Application No. PCT/US2009/046800, filed Jun. 9,
2009; which claims priority to U.S. Provisional Application No.
61/060,084, filed Jun. 9, 2008. The entire contents of each of the
above-referenced patents and patent applications are hereby
expressly incorporated by reference.
BACKGROUND
[0002] Cell therapy offers immense possibilities for treatment of a
wide variety of medical conditions. Currently cell therapy is
practiced in numerous embodiments, for example, bone marrow
transplantation for treatment of hematopoietic malignancies. The
successful establishment of procedures for transplantation of donor
cells into recipients whose own cells are malignant (leukemia),
altered (stroke, limb ischemia, etc.), or insufficient (due to
chemotherapy, radiotherapy, or congenital abnormality) constitutes
a major medical breakthrough in the therapeutic management of these
conditions.
[0003] One limiting factor of any cell therapy is the need for
blood-borne or directly injected cells to migrate to the targeted
tissue in order to maximize their therapeutic potential. With
regard to hematopoietic stem cells as a particular example, it is
known that only a small percentage of these cells home to the bone
marrow microenvironment when administered systemically. This
migration is regulated in part by adhesive factors present on the
luminal surface of endothelial cells that constitute the
microvascular lining of the bone marrow and in part by chemotactic
gradients secreted at a constant rate by bone marrow stromal cells.
In addition, for the treatment of myocardial infarction or stroke,
only a small fraction of injected stem cells actually home and
enter the area of tissue damage. Thus, there exists a need to
administer a high number of stem cells, sometimes prohibitively too
high to be obtained in an autologous or even allogeneic
setting.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawing(s) will be provided by
the Office upon request and payment of the necessary fee.
[0005] FIG. 1 illustrates the effect of pretreatment of human
neural stem cells (HNSCs) with .alpha.1,3 fucosyltransferase-VI
(FTVI) on the level of fucosylation. The expression of CLA was used
to determine the levels of fucosylation. The CLA expression by
hNSCs was determined by FACS analysis with untreated cells (A) or
following pre-incubation with the fucosylation mix (GDP-Fucose,
manganese and FTVI conditioned medium) (B). Isotype-matched IgG was
used as the negative control. Fluorescence intensity (FL2) of
samples was evaluated by FACSCanto.TM. cell analyzer (BD
Biosciences, San Jose, Calif.). The results of one experiment are
shown.
[0006] FIG. 2 graphically depicts the ex vivo expansion technique
utilized herein for T cells.
[0007] FIG. 3 contains a flow cytometry analysis of expanded
Regulatory T cells (Tregs).
[0008] FIG. 4 contains a flow cytometry analysis of Tregs
fucosylated with TZ101 (FTVI+GDP-fucose; Targazyme, Inc., Carlsbad,
Calif.).
[0009] FIG. 5 graphically illustrates the ability of TZ101 to
fucosylate various types of cells.
[0010] FIG. 6 graphically illustrates the ability of fucosylated
Tregs to prevent graft-versus-host disease (GVHD) in a mouse
model.
[0011] FIG. 7 graphically illustrates the ability of TZ102
(FTVII+GDP-fucose; Targazyme, Inc., Carlsbad, Calif.) to fucosylate
ex vivo expanded cytotoxic T cells (CTL).
[0012] FIG. 8 contains an analysis of the ability of FTVII-treated
CTL to kill leukemia cells in a xenogeneic AML mouse model.
DETAILED DESCRIPTION
[0013] Before explaining at least one embodiment of the inventive
concept(s) in detail by way of exemplary drawings, experimentation,
results, and laboratory procedures, it is to be understood that the
inventive concept(s) is not limited in its application to the
details of construction and the arrangement of the components set
forth in the following description or illustrated in the drawings,
experimentation, and/or results. The inventive concept(s) is
capable of other embodiments or of being practiced or carried out
in various ways. As such, the language used herein is intended to
be given the broadest possible scope and meaning; and the
embodiments are meant to be exemplary--not exhaustive. Also, it is
to be understood that the phraseology and terminology employed
herein is for the purpose of description and should not be regarded
as limiting.
[0014] Unless otherwise defined herein, scientific and technical
terms used in connection with the presently disclosed and claimed
inventive concept(s) shall have the meanings that are commonly
understood by those of ordinary skill in the art. Further, unless
otherwise required by context, singular terms shall include
pluralities and plural terms shall include the singular. Generally,
nomenclatures utilized in connection with, and techniques of, cell
and tissue culture, molecular biology, and protein and oligo- or
polynucleotide chemistry and hybridization described herein are
those well known and commonly used in the art. Standard techniques
are used for recombinant DNA, oligonucleotide synthesis, and tissue
culture and transformation (e.g., electroporation, lipofection).
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications or as commonly
accomplished in the art or as described herein. The foregoing
techniques and procedures are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification. See e.g., Sambrook
et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and
Coligan et al. Current Protocols in Immunology (Current Protocols,
Wiley Interscience (1994)), which are incorporated herein by
reference. The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0015] All patents, published patent applications, and non-patent
publications mentioned in the specification are indicative of the
level of skill of those skilled in the art to which this presently
disclosed and claimed inventive concept(s) pertains. All patents,
published patent applications, and non-patent publications
referenced in any portion of this application are herein expressly
incorporated by reference in their entirety to the same extent as
if each individual patent or publication was specifically and
individually indicated to be incorporated by reference.
[0016] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of the inventive concept(s) have been described in terms of
particular embodiments, it will be apparent to those of skill in
the art that variations may be applied to the compositions and/or
methods and in the steps or in the sequence of steps of the method
described herein without departing from the concept, spirit and
scope of the presently disclosed and claimed inventive concept(s).
All such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the inventive concept(s) as defined by the appended
claims.
[0017] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0018] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The singular
forms "a," "an," and "the" include plural referents unless the
context clearly indicates otherwise. Thus, for example, reference
to "a compound" may refer to 1 or more, 2 or more, 3 or more, 4 or
more or greater numbers of compounds. The term "plurality" refers
to "two or more." The use of the term "or" in the claims is used to
mean "and/or" unless explicitly indicated to refer to alternatives
only or the alternatives are mutually exclusive, although the
disclosure supports a definition that refers to only alternatives
and "and/or." Throughout this application, the term "about" is used
to indicate that a value includes the inherent variation of error
for the device, the method being employed to determine the value,
or the variation that exists among the study subjects. For example
but not by way of limitation, when the term "about" is utilized,
the designated value may vary by .+-.20% or .+-.10%, or .+-.5%, or
.+-.1%, or .+-.0.1% from the specified value, as such variations
are appropriate to perform the disclosed methods and as understood
by persons having ordinary skill in the art. The use of the term
"at least one" will be understood to include one as well as any
quantity more than one, including but not limited to, 2, 3, 4, 5,
10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may
extend up to 100 or 1000 or more, depending on the term to which it
is attached; in addition, the quantities of 100/1000 are not to be
considered limiting, as higher limits may also produce satisfactory
results. In addition, the use of the term "at least one of X, Y and
Z" will be understood to include X alone, Y alone, and Z alone, as
well as any combination of X, Y and Z. The use of ordinal number
terminology (i.e., "first", "second", "third", "fourth", etc.) is
solely for the purpose of differentiating between two or more items
and is not meant to imply any sequence or order or importance to
one item over another or any order of addition, for example.
[0019] As used in this specification and claim(s), the terms
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0020] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, CBA,
BCA, ACB, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and
so forth. The skilled artisan will understand that typically there
is no limit on the number of items or terms in any combination,
unless otherwise apparent from the context.
[0021] As used herein, the term "substantially" means that the
subsequently described event or circumstance completely occurs or
that the subsequently described event or circumstance occurs to a
great extent or degree. For example, the term "substantially" means
that the subsequently described event or circumstance occurs at
least 90% of the time, or at least 95% of the time, or at least 98%
of the time.
[0022] The ability of cells such as leukocytes to interact with the
endothelium has been known for decades. It has also been known that
various glycosylation patterns are critical for cells such as
leukocytes to "roll" on the endothelium prior to extravasation.
What is desirable is the identification of novel methods that
enhance cell trafficking and engraftment to areas of need in a
simple and clinically applicable manner.
[0023] The present embodiments relate generally to the field of
cell therapy. More specifically, some embodiments relate to methods
of enhancing the natural process of cell migration through
augmentation of specific glycosylation features on the surface of
various cell types. More specifically, some embodiments relate to
treatment of cells with fucosyltransferases in order to enhance the
interaction between blood-borne stem cells, progenitor cells and
endothelial cells facilitating entry into biological niches and
tissues where they may function on a number of different levels for
therapeutic and restorative intervention.
[0024] Accordingly, provided herein in certain embodiments are
methods of enhancing homing and engraftment of
therapeutically-administered cells in a patient. It should be noted
that the term "patient" is meant to broadly include any animal. For
example, the animal can be a mammal, a bird, a fish, a reptile, a
fish, an insect or any other animal. Some non-limiting examples of
mammals may include humans and other primates, equines such as
horses, bovines such as cows, mice, rats, rabbits, Guinea Pigs,
pigs, and the like. It is also worth noting that the compositions
and methods can be used with or applied to individual cells (for
example ex vivo treatment or modification), to insect cells, etc.
Also provided are cells that have been modified to enhance homing
and engraftment. The embodiments provided herein are based in part
on the surprising finding that by modification of molecules
involved in the cell-endothelium interaction, it is possible to
enhance the homing and subsequent efficacy of cell therapy.
[0025] One embodiment provides a method of enhancing homing and
engraftment of a therapeutically-administered cell in a patient in
need of treatment with a cell population; providing cells that may
have been contacted with an agent that modifies at least one
surface molecule on the cells, resulting in a population of
modified cells; and providing or administering the population of
modified cells to a patient in need thereof. In certain aspects,
the cell surface molecule may be modified so as to result in an
alteration of cell charge.
[0026] In one embodiment, a method of enhancing homing and
engraftment of a cell may comprise providing one or more cells
selected from stem cells, progenitor cells, neutrophils,
macrophages, T-cells, and combinations thereof. The stem or
progenitor cells may be embryonic stem cells, adult stem cells,
expanded stem cells, placental stem cells, bone marrow stem cells,
amniotic fluid stem cells, neuronal stem cells, cardiomyocyte stem
cells, placental stem cells, endothelial progenitor cells,
circulating and mobilized peripheral blood stem cells, muscle stem
cells, germinal stem cells, adipose tissue derived stem cells,
exfoliated teeth derived stem cells, hair follicle stem cells,
dermal stem cells, parthenogenically derived stem cells,
reprogrammed stem cells such as induced pluripotent stem cells, or
somatic nuclear transfer and side population stem cells, as well as
any combination thereof. One or more cells may have been contacted
with an agent that modifies at least one surface molecule on the
cell that may result in enhanced selectin-mediated binding. This
may result in a population of modified cells. These cells may be
provided to animals. Such animals may include birds, reptiles,
fish, insects, and mammals including but not limited to humans,
equines such as horses, bovines such as cows, dogs, mice, rats,
pigs, guinea pigs, rabbits and the like.
[0027] In certain aspects of the above embodiments, the cell
surface molecule may be modified by treatment with an enzyme and
appropriate substrate(s) under conditions sufficient for causing an
alteration of cell surface charge. In certain aspects, the enzyme
may be a glycosidase, glycosyltransferase, a fucosyltransferase, a
neuraminidase, an acetylglucosaminyltransferase, or any
glycosyltransferase capable of increasing the number or affinity of
cell surface selectin binding components. In certain aspects, the
enzyme may be alpha 1,3-fucosyltransferase I, alpha
1,3-fucosyltransferase III, alpha 1,3-fucosyltransferase IV, alpha
1,3-fucosyltransferase V, alpha 1,3-fucosyltransferase VI, alpha
1,3-fucosyltransferase VII, or alpha 1,3-fucosyltransferase IX.
[0028] In another aspect of the above embodiment, the cell may be
treated with a reagent or reagents that link a binding unit to the
cell surface. The binding unit may consist of a particle as well as
a ligand of natural or non-natural sugars shown to possess binding
affinity for receptors present on endothelial cells similar to that
seen with natural sugars. The added binding unit may increase the
functionalization of the cell.
[0029] In certain further aspects of the above embodiment, the cell
may be treated with a single or plurality of molecules having
ability to cause alpha 1-3 fucosylation of glycan determinants. In
certain aspects, the molecule may be an alpha 1-3
fucosyltransferase mixed together with a concentration of a fucose
carrier under conditions sufficient to provide enhanced alpha 1-3
fucosylation of glycan determinants. In certain aspects, the fucose
carrier may be guanosine diphosphate fucose. In certain aspects,
the alpha 1-3 fucosyltransferase may be alpha 1-3
fucosyltransferase VI. In other aspects, the alpha 1-3
fucosyltransferase may be alpha 1-3 fucosyltransferase VII. In
other aspects, the alpha 1-3 fucosyltransferase may be alpha 1-3
fucosyltransferase IV.
[0030] In certain aspects of the above embodiment, prior to the
providing or administering, the population of modified cells has
been further contacted for a period of time insufficient for cell
division to occur with a CD26 peptidase inhibitor in an amount
effective to inhibit CD26 peptidase activity and effective to
increase the migratory response to CXCL12. PCT Publication WO
2009/152186, which is incorporated herein by reference in its
entirety, discloses and describes methods and compositions, any of
which can be used with the technology of this application in any
combination.
[0031] In certain aspects, prior to providing modified cells, a
recipient may be contacted for a period of time and with sufficient
dosing of a CD26 peptidase inhibitor in an amount effective to
inhibit recipient CD26 peptidase activity effective to increase the
migratory response of donor cells to chemotractant agents such as
stromal cell-derived factor.
[0032] In certain aspects, the cell population may comprise or
consist essentially of a population of stem cells, both embryonic
and adult and expanded cell populations. In certain aspects, the
stem cells may be embryonic stem cells, cord blood stem cells,
placental stem cells, bone marrow stem cells, amniotic fluid stem
cells, hematopoietic stem cells, mesenchymal stem cells, neuronal
stem cells, cardiomyocyte stem cells, circulating and immobilized
peripheral blood stem cells, endothelial progenitor cells,
monocyte-derived stem cells, muscle stem cells, germinal stem
cells, adipose tissue derived stem cells, exfoliated teeth derived
stem cells, hair follicle stem cells, dermal stem cells,
parthenogenically derived stem cells, reprogrammed stem cells such
as induced pluripotent stem cells or somatic nuclear transfer and
side population stem cells. In certain aspects, the embryonic stem
cells may be totipotent. In certain aspects, the stem cell may be
hematopoietic, mesenchymal, neural or cardiomyocyte stem cells. In
certain aspects the hematopoietic stem cells may be further defined
and differentiated as CD38-, lin- or ALDH-bright cells.
[0033] In certain aspects, the cell population may comprise or
consist essentially of a population of committed progenitor cells
or differentiated cells. In certain aspects, the cell population
may be a mature blood cell population. In certain aspects, the
mature blood cell may be neutrophils, macrophages, T-cells,
activated T-cells, helper T cells, cytolytic T-cells, memory
T-cells, regulatory T-cells, natural killer (NK) cells, or
reprogrammed cells. In certain aspects, the T-cells may be from a
heterogeneous population of T-cells.
[0034] In certain aspects, the patient in need of treatment with a
cell population suffers from a malignant or non-malignant blood
disorder such as an acute leukemia, a chronic leukemia, a
myelodysplastic syndrome, a stem cell disorder, a
myeloproliferative disorder, a lymphoproliferative disorder, a
phagocyte disorder, a histiocytic disorder, a lysosomal storage
disease, an age related disorder, an arterial or blood vessel or
cardiovascular disorder, an enzyme deficiency disorder, a
congenital immune system disorder, an inherited erythrocyte
abnormality, an inherited platelet abnormality, a plasma cell
disorder, a tumor or an autoimmune disease. In certain aspects, the
patient in need of treatment with a cell population may suffer from
peripheral arterial diseases, ischemic limb injury, diabetes, heart
disease, bone disease, liver disease, muscular dystrophy,
Alzheimer's disease, ALS, multiple sclerosis, Parkinson's disease,
spinal cord injury, stroke or infertility.
[0035] In certain aspects, the population of modified cells may be
administered intravenously, intraarterially, intramuscularly,
subcutaneously, transdermally, intratracheally, intraperitoneally,
intrathecally intracranially, intravitreally, or directly into the
microvascular compartment of bone or into spinal fluid. In certain
aspects, the population of modified cells may be administered in or
proximal to a site of injury. In certain aspects, the homing and
engraftment may take place within the bone marrow of the patient in
need thereof.
[0036] In another embodiment, a composition may comprise an
isolated population of cells modified for enhanced
selectin-mediated binding. The isolated population of cells may be
neutrophils, macrophages, T-cells, subpopulation of T-cells, or
stem or progenitor cells selected from a group consisting of:
embryonic stem cells, adult stem cells, expanded stem cells,
placental stem cells, bone marrow stem cells, amniotic fluid stem
cells, neuronal stem cells, cardiomyocyte stem cells, endothelial
progenitor cells, circulating and mobilized peripheral blood stem
cells, muscle stem cells, germinal stem cells, adipose tissue
derived stem cells, exfoliated teeth derived stem cells, hair
follicle stem cells, dermal stem cells, parthenogenically derived
stem cells, or reprogrammed stem cells such as induced pluripotent
stem cells or somatic nuclear transfer and side population stem
cells and a pharmaceutically-acceptable carrier.
[0037] In certain aspects, the isolated population may comprise a
cell surface modification. In certain aspects, the cell surface
molecule may be modified by treatment with an enzyme and
appropriate substrate(s) under conditions sufficient for causing an
alteration of cell surface charge.
[0038] In certain aspects, the enzyme is selected from a group
comprising of: a glycosidase, a glycosyltransferase, a
fucosyltransferase, a neuraminidase, and an
acetylglucosaminyltransferase or any other glycotransferases
capable of increasing cell surface selectin binding components. In
certain aspects, the enzyme is selected from a group comprising of
alpha 1,3-fucosyltransferase I, alpha 1,3-fucosyltransferase III,
alpha 1,3-fucosyltransferase IV, alpha 1,3-fucosyltransferase V,
alpha 1,3-fucosyltransferase VI, alpha 1,3-fucosyltransferase VII
and alpha 1,3-fucosyltransferase IX.
[0039] In certain aspects, the cell may be treated with a single or
plurality of molecules having ability to cause alpha 1-3
fucosylation of glycan determinants. In certain aspects, the
molecule may be an alpha 1-3 fucosyltransferase mixed together with
a concentration of a fucose carrier under conditions sufficient to
provide enhanced alpha 1-3 fucosylation of glycan determinants. In
certain aspects, the fucose carrier may be guanosine diphosphate
fucose. In certain aspects, the alpha 1-3 fucosyltransferase may be
alpha 1-3 fucosyltransferase VI. In certain aspects, the alpha 1-3
fucosyltransferase may be alpha 1-3 fucosyltransferase VII. In
certain aspects the alpha 1-3 fucosyltransferase may be alpha 1-3
fucosyltransferase IV. In certain aspects the molecule may be a
non-naturally occurring enzyme having the ability to add a glycan
determinant or a non-natural sugar that mimics the activity of
fucose or other sugars that enhance the selectin binding
process.
[0040] In certain aspects, the cell population may comprise or
consist essentially of a population of stem cells both embryonic
and adult. In certain aspects, the stem cells may be embryonic stem
cells, cord blood stem cells, placental stem cells, bone marrow
stem cells, amniotic fluid stem cells, hematopoietic stem cells,
mesenchymal stem cells, neuronal stem cells, cardiomyocyte stem
cells, circulating and mobilized peripheral blood stem cells,
endothelial progenitor cells, monocyte-derived stem cells, muscle
stem cells, germinal stem cells, adipose tissue derived stem cells,
exfoliated teeth derived stem cells, hair follicle stem cells,
dermal stem cells, parthenogenically derived stem cells,
reprogrammed stem cells such as induced pluripotent stem cells or
somatic nuclear transfer and side population stem cells. In certain
aspects, the embryonic stem cells may be totipotent. In certain
aspects, the stem cell may be hematopoietic, mesenchymal, neural or
cardiomyocyte stem cells.
[0041] In certain aspects, the cell may be a mature blood cell. In
certain aspects, the mature blood cell may be a neutrophil,
macrophage, or T-cell. In certain aspects, the T-cells may be from
a heterogeneous population of T-cells or from an ex vivo expanded
cell population.
[0042] Another embodiment provides a method of enhancing homing and
engraftment of a cell, comprising providing one or more cells
selected from stem cells, progenitor cells, neutrophils,
macrophages and T-cells. The stem or progenitor cells may be
embryonic stem cells, adult stem cells, expanded stem cells,
placental stem cells, bone marrow stem cells, amniotic fluid stem
cells, neuronal stem cells, cardiomyocyte stem cells, endothelial
progenitor cells, circulating and immobilized peripheral blood stem
cells, muscle stem cells, germinal stem cells, adipose tissue
derived stem cells, exfoliated teeth derived stem cells, hair
follicle stem cells, dermal stem cells, parthenogenically derived
stem cells, reprogrammed stem cells such as induced pluripotent
stem cells or somatic nuclear transfer and side population stem
cells. One or more cells may be contacted with an agent that
modifies at least one surface molecule on the cell(s) to result in
enhanced selectin-mediated binding, resulting in a population of
modified cells.
[0043] Also provided herein is a method of fucosylation of cells so
as to increase the ability of the cells to traffic, home and
engraft into an area of biological need. The cells may be mature
fully differentiated cells whose homing to specific targets is
desired, such as islets, hepatocytes, or neutrophils or cells may
be progenitor cells capable of differentiating into functional
cells such as hepatic, renal, cardiac, or islet progenitors, or
alternatively, the cells may be stem cells with multilineage
differentiation ability such as embryonic stem cells, cord blood
stem cells, placental stem cells, bone marrow stem cells, amniotic
fluid stem cells, neuronal stem cells, circulating and mobilized
peripheral blood stem cells, mesenchymal stem cells, endothelial
stem cells, cardiomyocyte stem cells, germinal stem cells,
committed endothelial progenitor cells, committed progenitor cells,
adipose tissue derived stem cells, exfoliated teeth derived stem
cells, hair follicle stem cells, dermal stem cells,
parthenogenically derived stem cells, chemically, biologically, or
electronically reprogrammed stem cells such as induced pluripotent
stem cells or somatic nuclear transfer and side population of stem
cells.
[0044] Other aspects relate to enhancing the ability of cells to
modulate the immune system through enabling the cells to function
with augmented efficiency at trafficking and homing. In another
aspect, cells useful for immune therapy are "reprogrammed" ex vivo
with endowment of distinct immunological properties. Surface
modification may be performed before reprogramming, during
reprogramming or after reprogramming. Reprogramming may be
performed so as to increase immune stimulatory properties of the
immune cells, or may be performed to allow the immune cells to
suppress other immune cells. Reprogramming may be performed during
expansion of cells, or to cells that have already been
expanded.
[0045] In certain aspects, the cells can be fucosylated so as to
enhance ability to home. Fucosylation may be performed on specific
molecules present on the cells, or may be performed globally in a
non-specific manner. Cells may be fucosylated through culture with
an enzyme such as a fucosyltransferase capable of transferring
fucose groups such as a fucosyltransferase. In certain aspects, the
fucosyltransferase may be an alpha 1,3 fucosyltransferase. In
another aspect the enzyme is selected from a group comprising of
alpha 1,3-fucosyltransferase IX, alpha 1,3-fucosyltransferase III,
alpha 1,3-fucosyltransferase IV, alpha 1,3-fucosyltransferase V,
alpha 1,3-fucosyltransferase VI and alpha 1,3-fucosyltransferase
VII. Appropriate culture conditions and substrates are also
provided within the scope of the embodiments in order to allow
proper fucosylation to occur. The conditions may include addition
of substrates such as GDP-fucose or other similar compounds that
provide a source of fucose.
[0046] In certain aspects, cells may be treated with a single or
plurality of agents in order to augment expression of proteins
involved in migration. Since the proteins involved in migration,
when expressed de-novo, are not properly fucosylated, the addition
of exogenous fucose groups increases ability of the de-novo
expressed proteins to interact with endothelium and properly home.
Specific molecules may include histone deacetylase inhibitors or
DNA methyltransferase inhibitors.
[0047] Some embodiments relate generally to compositions for and
methods of enhancing homing and engraftment of a
therapeutically-administered cell in a patient. Also, some
embodiments relate to cells that have been modified to enhance
homing and engraftment. The embodiments provided herein are based
in part on the surprising finding that by modification of molecules
involved in the cell-endothelium interaction, it is possible to
enhance the homing and subsequent efficacy of cell therapy.
[0048] The process by which cells exit the systemic circulation and
enter distinct biologically niches is a complex coordinated process
that involves numerous molecules. The process of cellular exit is a
bi-directional communication between the vascular endothelium cells
and the circulating stem and progenitor cells. This process has
been best characterized in the description of leukocyte exit from
systemic circulation. An important family of molecules involved
critically and initially in the process of cellular trafficking is
the selectins. These molecules are type 1 transmembrane proteins
that contain what is known as C-type lectin domains. Lectins are
proteins that bind sugars. Most commonly known lectins include
conconavalin-A and phytohemagglutinin. The C-type lectin domains on
the selectins reside at the N-terminal of the selectins and
interact with a wide array of glycoprotein ligands. Since the
lectin domains bind sugar moieties, it is important that proper
placement of sugars onto the proteins such that interaction of the
selectins occurs. Since placement of sugars (glycosylation) usually
occurs as a post-translational event, the mere genetic manipulation
of cells is not sufficient usually to alter ability of cells to
interact with selectins. The exception to this is, of course,
genetic manipulation in the sense of transfecting cells with
enzymes that are involved in the addition of sugars.
[0049] Provided herein are means of endowing enhanced
trafficking/homing capabilities onto cells for use in cell therapy,
said means consisting of modification of various glycosylation
patterns on cells in order to augment ability to "roll", "tether",
and "adhere" on the endothelium. The concept of cells rolling,
tethering, and adhering on the endothelium is commonly known in the
art and means of augmentation of this rolling process have been
described strictly in the areas of hematopoietic cells, as well as
tumor cells.
[0050] Previous studies with hematopoietic cells have demonstrated
that it is possible to alter glycosylation and fucosylation
patterns on the surface of cells by treating of cells with enzymes
such as fucosyltransferases. In addition to establishing the
ability to modify the surface of cells, the functional consequences
of this modification have been documented. Specifically, several
reports (Xia et al., Blood 2004 104:3091-3096; Hidalgo, et al., J.
Clin. Invest. 2002 110:559-569, each of which is hereby
incorporated by reference in its entirety) demonstrated that
fucosylation of cord blood hematopoietic cells enhances binding of
P and E selectin, enhances ex vivo binding to P and
E-selectin-coated plates under physiological shear stress
conditions and enhances homing and engraftment into bone marrow of
NOD-SCID mice. (See also U.S. Pat. No. 7,332,334 and US Pub. No.
2006/0228340 to Xia and McEver, each of which is hereby
incorporated by reference in its entirety). Findings in mesenchymal
stem cells have also been described by others, including Sackstein
et al. (US 2003/0040607 and US 2008/0044383, each of which is
incorporated by reference in its entirety). Each reference listed
in this paragraph is incorporated herein by reference in its
entirety.
[0051] However another report demonstrated that while early
(minutes to hours following iv injection) adhesion of cord blood
hematopoietic cells is increased after ex vivo fucosylation, no
increase in bone marrow homing at 16-24 hrs was observed (Hidalgo
and Frenette, Blood 2005 105:567-575, which is incorporated herein
by reference in its entirety).
[0052] It has been suggested by Sackstein et al. that mesenchymal
stem cells, which do not express E-selectin ligands, can be
glycosylated enzymatically in an effort to enhance migration of
these cells to the bone marrow (Sackstein et al. Nature Medicine
2008 14:181-187; which is incorporated herein by reference in its
entirety).
[0053] Thus, embodiments presented herein are based in part on the
novel observation that fucosylation of ligands increases binding to
tissue and can be used to enhance migration of various types of
cells, enumerated herein, to an area of need. Specifically, some
embodiments provided herein relate in part to the surprising
finding that augmentation of the tethering and rolling process
through various means is useful for enhancing functional
capabilities of a wide variety of non-hematopoietic cells and stem
cells. Accordingly, the methods and compositions provided herein
may be useful for treatment of a wide variety of medical conditions
that are amenable to cell therapy.
[0054] Therapeutic Methods
[0055] In accordance with the above, provided herein are methods of
enhancing homing and engraftment of a therapeutically-administered
cell in a patient. Specifically, provided herein are methods of
modification of sugar residues, both natural and non-natural, on
the surface of cells used for cell therapy so as to enhance their
interaction with members of the selectin family, thereby enhancing
trafficking of the cells administered systemically to an area of
need.
[0056] Also provided are cells that have been modified to enhance
homing and engraftment. The embodiments provided herein are based
in part on the surprising finding that by modification of molecules
involved in the cell-endothelium interaction, it is possible to
enhance the homing and subsequent efficacy of cell therapy.
[0057] Examples of cells include, without being limited thereto,
neutrophils, macrophages and T-cells, wherein the stem or
progenitor cells are selected from a group consisting of: embryonic
stem cells, adult stem cells, expanded stem cells, placental stem
cells, bone marrow stem cells, hematopoietic stem cells,
mesenchymal stem cells, amniotic fluid stem cells, neuronal stem
cells, cardiomyocyte stem cells, endothelial progenitor cells,
circulating and mobilized peripheral blood stem cells, muscle stem
cells, germinal stem cells, adipose tissue derived stem cells,
exfoliated teeth derived stem cells, hair follicle stem cells,
dermal stem cells, parthenogenically derived stem cells,
reprogrammed stem cells such as induced pluripotent stem cells or
somatic nuclear transfer and side population stem cells. In some
aspects, one or more of the cell types mentioned can be
specifically excluded from the methods or compositions described
herein. As one example, in some aspects mesenchymal or
hematopoietic stem cells can be excluded.
[0058] In one embodiment is provided a method of enhancing homing
and engraftment of a therapeutically-administered cell in a patient
comprising selecting a patient in need of treatment with a cell
population; providing cells that have been contacted with an agent
that modifies at least one surface molecule on the cells, resulting
in a population of modified cells; and providing or administering
the population of modified cells to a patient in need thereof. In
certain aspects, the cell surface molecule is modified so as to
result in an alteration of cell charge.
[0059] Modification Enzymes
[0060] In certain aspects of the above embodiments, the cell
surface molecule is modified by treatment with an enzyme and
appropriate substrate(s) under conditions sufficient for causing an
alteration of cell surface charge. Enyzmes that modify cell surface
molecules are known in the art. Such enzymes include a purified
glycosyltransferase polypeptide. Glycosyltransferase include for
example, fucosyltransferase, galactosyltransferase,
sialytransferase and N-acetylglucosaminotransferase. The
fucosyltransferase can be, for example, an alpha 1,3
fucosyltransferase such as an alpha 1,3-fucosyltransferase I, alpha
1,3-fucosyltransferase III, alpha 1,3-fucosyltransferase IV, alpha
1,3-fucosyltransferase V, alpha 1,3-fucosyltransferase VI, alpha
1,3-fucosyltransferase VII, and alpha 1,3-fucosyltransferase IX. It
should be noted that in some embodiments, one or more of the
enzymes listed herein can be specifically excluded. For example, in
some aspects, FTVI can be specifically excluded from the methods
and compositions described herein.
[0061] In certain aspects, the cell surface molecule is modified in
the presence of a sugar donor suitable for the specific
glycosyltransferase. Sugar donors for glycosyltransferases are
known in the art. For example, when the glycoslytransferase is a
fucosyltransferase, the donor is GDP-fucose. Whereas, when the
glycosyltransferase is a siayltransferase, the donor is eMP-sialic
acid. In some instances the sugar can be a nonnatural sugar added
by a natural or modified glycosyltransferase.
[0062] The glycosyltransferases are biologically active. By
biologically active is meant that the glycosyltransferases are
capable of transferring a sugar molecule from a donor to acceptor.
For example, the glycosyltransferase is capable of transferring
0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 5, 10 or more
.mu.moles of sugar per minute at pH 6.5 at 37.degree. C.
[0063] Physiologically acceptable solution is any solution that
does not cause cell damage, e.g. death. For example, the viability
of the cell or cell particle is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99% or more after treatment according to the methods
presented herein. Suitable physiologically acceptable solutions
include for example, Hank's Balanced Salt Solution (HBSS),
Dulbecco's Modified Eagle Medium (DMEM), a Good's buffer (see N. E.
Good, G. D. Winget, W. Winter, T. N. Conolly, S. Izawa and R. M. M.
Singh, Biochemistry 5, 467 (1966); N. E. Good, S. Izawa, Methods
Enzymol. 24, 62 (1972) such as a HEPES buffer, a
2-Morpholinoethanesulfonic acid (MES) buffer, or phosphate buffered
saline (PBS). Each reference listed in this paragraph is
incorporated herein by reference in its entirety.
[0064] Thus, in certain embodiments provided herein, cells can be
treated ex vivo with a kit that contains some or all of the agents,
such as enzyme, buffer, cofactors and substrate necessary to
achieve fucosylation of the cell surface glycoproteins that mediate
adhesive interactions between the circulating cells following iv
administration and endothelial cells at targeted tissue sites. In
another embodiment provided herein, cells such as cord blood can be
pretreated with the fucosylation kit prior to freezing or
storage.
[0065] In certain aspects, the syn-anti and/or .alpha.-.beta.
substitution and orientation of the polysaccharide are modulated to
provide the desired effect. Cell surface oligosaccharides are
highly diversified in their structures and are associated with a
variety of cell functions. In an inflammatory response, for
example, neutrophils or leukocytes bind to injured tissues where
the adhesion process occurs. This process has been found to be
mediated by the tetrasaccharide sialyl Lewis X on neutrophiles or
leukocytes and the receptor ELAM-1 (endothelial leukocyte adhesion
molecule 1), a glycoprotein of the selectin family. Several sialyl
lewis analogues and mimetics have been analyzed, in part, to
understand affects of syn versus anti sugar conformers. Similar
studies have examined fucosyl and galactosyl conformers,
diastereomers, epimers and chiral analogues to examine adhesion and
inhibitory properties. See Ichikawa Y et al. J. Am. Chem. Soc.
1992, 114, 9283-9298; Nelson, Richard M. et al J. Clin. Invest.
1993, 1157-1166; Chun-Cheng Lin et al, J. Am. Chem. Soc. 1996, 118,
6826-6840; Clarke Julia L. J Am. Chem. Soc. 1996, 118, 6826-6840;
Chikara Ohyama, et al, The EMBO Journal Vol. 18 No. 6 pp.
1516-1525, 1999; each of which is incorporated herein by reference
in its entirety. Each reference listed in this paragraph is
incorporated herein by reference in its entirety.
[0066] The introduction of fucose onto surface glycans of the cells
of interest can be accomplished by enzymatic transfer from a donor
substrate utilizing an alpha 1-3-fucosyltransferase (FT) by a
process well known to someone skilled in the art. For this transfer
to occur the cells at varying concentrations can be exposed to an
incubation buffer containing a number of ingredients each of which
can be optimized for efficient transfer of the fucose. The
selection of buffer can come from a number of available buffers
with Hanks balanced salt solution (HBSS) serving as the primary
example. The substrate, guanosine diphosphate-fucose (GDP-fucose),
at 1 mM can be mixed with the FT added at sufficient activity,
expressed as Units/mL, to achieve maximal transfer of fucose to the
cells of interest. In addition, MnC12 at a final concentration of
0-10 mM can be added, if needed, depending on the cell population
to further accelerate the enzymatic transfer reaction. The
temperature and time of incubation can also be optimized for
maximal transfer of fucose under practical application conditions
with minimum toxicity to the cells of interest but is generally
conducted at 37.degree. C. for 40 minutes.
[0067] Confirmation of fucosylated epitopes on the cells of
interest as means of confirming maximal levels of fucosylation can
be verified by Flow Cytometry utilizing agents and procedures well
known to someone skilled in the art. For example, sialyl LewisX is
a fucosylation epitope found on both P and E-selectins. By
incubation of the FT-treated cells with anti-sLeX mAb HECA 452
(IgM) followed by treatment with FITC-conjugated fragment to the
IgM, the sLeX epitopes on the cell surface can be visualized using
standard Flow Cytometry procedures.
[0068] Combination Treatment with CD26 Peptidase Inhibitors
[0069] In certain aspects of the above embodiment, prior to the
administering, the population of modified cells has been further
contacted for a period of time insufficient for cell division to
occur with a CD26 peptidase inhibitor in an amount effective to
inhibit CD26 peptidase activity and effective to increase the
migratory response to CXCL12.
[0070] Exemplary methods of treating stem cells with CD26
(dipeptidylpeptidase) inhibitors are described in Christopherson et
al. (US Pub No. 2004/0247574, incorporated by reference in its
entirety). In certain aspects, the CD26 inhibitor is selected from
the group consisting of Diprotin A (Ile-Pro-Ile),
Valine-Pyrrolidide, sitagliptin, vildagliptin, saxagliptin,
alogliptin or any other class of compounds shown to exhibit potent
inhibition of either purified, soluble or cell surface (CD26)
dipeptidylpeptiase. PCT Publication WO 2009/152186, which is
incorporated herein by reference in its entirety, discloses and
describes methods and compositions, any of which can be used with
the technology of this application in any combination.
[0071] In certain aspects, the cell population is contacted with
said CD26 inhibitor for about 5 minutes to about 12 hours or
conditions suitable for sufficient inhibition of cell surface CD26
leading to an enhanced migratory response to chemotactic factors
such as stromal cell-derived factor. In certain aspects, the cell
population is contacted with said CD26 inhibitor for about 5
minutes to about 12 hours. In certain aspects, the cell population
is contacted with said CD26 inhibitor for less than 6 hours. In
certain aspects, the cell population is contacted with said CD26
inhibitor for less than 2 hours. In certain aspects, the cell
population is contacted with said CD26 inhibitor for less than 1
hour.
[0072] In certain aspects, the inhibitor is administered in a
concentration of less than about 1 nM, about 1, .mu.M, about 5,
.mu.M, about 10, .mu.M, about 50, .mu.M, about 100, .mu.M, about 1
mM or about 5 mM. In certain aspects, the inhibitor is administered
in a concentration of no less than about 5 mM.
[0073] In certain aspects, at least 1 donor cell is treated. In
selected embodiments, at least 1.times.10.sup.2, 1.times.10.sup.3,
1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8 donor cells per mL are
treated.
[0074] In certain aspects the recipient can be treated with CD26
inhibitor simultaneous with injection of cell surface modified
cells. In certain aspects the recipient can be treated with CD26
inhibitor prior to injection of cell surface modified cells. In
certain aspects the recipient is pretreated with single or multiple
doses of the CD26 inhibitor either simultaneous or prior to cell
injection to achieve sustained inhibition of either or both
administered cells and recipient CD26 activity leading to enhanced
homing of administered cells.
[0075] Therapeutic Cell Populations
[0076] Stem Cells. In certain aspects, the cell population
comprises or consists essentially of a population of stem cells. In
certain aspects, the stem cells are selected from a group
consisting of: embryonic stem cells, cord blood stem cells,
placental stem cells, bone marrow stem cells, amniotic fluid stem
cells, hematopoietic stem cells, mesenchymal stem cells, neuronal
stem cells, cardiomyocyte stem cells, circulating and immobilized
peripheral blood stem cells, mesenchymal stem cells, germinal stem
cells, adipose tissue derived stem cells, exfoliated teeth derived
stem cells, hair follicle stem cells, dermal stem cells,
parthenogenically derived stem cells, reprogrammed stem cells such
as induced pluripotent stem cells or somatic nuclear transfer and
side population stem cells or transdifferentiated cells. In certain
aspects, the embryonic stem cells are totipotent.
[0077] As used herein, a "mesenchymal cell" means a cell forming a
mesenchymal tissue, such as osteoblast, chondrocyte, myoblast,
adipocyte, stroma cell, tendon cell, and the like, a mesenchymal
stem cell capable of differentiating into these cells, and its
premesenchymal stem cell. Mesenchymal cells generated during the
embryo development, mesenchymal cells within an animal body, and
mesenchymal cells differentiated and generated from pluripotent
stem cells in vitro or in vivo are all encompassed in the term
"mesenchymal cell."
[0078] As used herein, a "mesenchymal stem cell" means a
mesenchymal cell possessing the ability of differentiating into
mesenchymal cells of one or more types and the ability of
self-replication. The mesenchymal stem cell differentiated from a
pluripotent stem cell in vitro is positive for PDGFR.alpha. and
negative for FLK1. Mesenchymal stem cells are able to differentiate
into osteoblasts, chondrocytes, myoblasts, adipocytes, stroma
cells, tendon cells, and the like, as with mesodermal cells.
[0079] As used herein, a "premesenchymal stem cell" means a
mesenchymal cell possessing the ability of differentiating into
mesenchymal stem cells of one or more types and the ability of
self-replication. The premesenchymal stem cell differentiated from
a pluripotent stem cell in vitro expresses Sox1, a neuroectodermal
marker. The premesenchymal stem cell is able to differentiate into
a mesenchymal stem cell which is PDGFR.alpha.-positive and
FLK1-negative.
[0080] As used herein, a "neural stem cell" refers to a multipotent
cell obtained from the central nervous system that can be caused to
differentiate into cells that posses one or more biological
activities of a neuronal cell type. Neural stem cells differentiate
into neurons, astrocytes, and oligodendrocytes after plating onto
substrates which stimulate adhesion and differentiation, for
example poly-L-ornithine orlaminin. In addition, these multipotent
CNS stem cells proliferate and expand in response to epidermal
growth factor ("EGF") and basic fibroblast growth factor ("bFGF")
and differentiate into neurons, astrocytes, oligodendrocytes, and
muscle stem cells.
[0081] Committed Progenitor Cells and Differentiated Cells
[0082] In certain aspects, the cell population comprises or
consists essentially of a population of committed progenitor cells
or differentiated cells or transdifferentiated cells. In certain
aspects, the cell population is a mature blood cell population. In
certain aspects, the mature blood cell is selected from the group
consisting of: neutrophils, macrophages and T-cells. In certain
aspects, the T-cells are from a heterogeneous population of
T-cells.
[0083] Patients in Need of Treatment with Modified Cell
Populations
[0084] In certain aspects, the patient in need of treatment with a
cell population suffers from a condition selected from the group
consisting of: an acute leukemia, a chronic leukemia, a
myelodysplastic syndrome, a stem cell disorder, a
myeloproliferative disorder, a lymphoproliferative disorder, a
phagocyte disorder, a histiocytic disorder, a lysosomal storage
disease, a congenital immune system disorder, an inherited
erythrocyte abnormality, an inherited platelet abnormality, a
plasma cell disorder, a tumor and an autoimmune disease. In certain
aspects, the patient in need of treatment with a cell population
suffers from a condition selected from the group consisting of:
peripheral arterial diseases, ischemic limb injury, diabetes, heart
disease, liver disease, bone disease, muscular dystrophy,
Alzheimer's disease, ALS, multiple schlerosis, Parkinson's disease,
spinal cord injury, stroke and infertility. Further examples of the
above-described conditions are set forth in Table I below.
TABLE-US-00001 TABLE I Acute Leukemias Chronic Leukemias Acute
Biphenotypic Leukemia Chronic Lymphocytic Leukemia (CLL) Acute
Lymphocytic Leukemia (ALL) Chronic Myelogenous Leukemia (CML) Acute
Myelogenous Leukemia (AML) Juvenile Chronic Myelogenous Leukemia
Acute Undifferentiated Leukemia (JCML) Juvenile Myelomonocytic
Leukemia (JMML) Myelodysplastic Syndromes Stem Cell Disorders
Amyloidosis Aplastic Anemia (Severe) Chronic Myelomonocytic
Leukemia Congenital Cytopenia (CMML) Dyskeratosis Congenita
Refractory Anemia (RA) Fanconi Anemia Refractory Anemia with Excess
Blasts Paroxysmal Nocturnal Hemoglobinuria (PNH) (RAEB) Refractory
Anemia with Excess Blasts in Transformation (RAEB-T) Refractory
Anemia with Ringed Sideroblasts (RARS) Myeloproliferative Disorders
Lymphoproliferative Disorders Acute Myelofibrosis Hodgkin's Disease
Agnogenic Myeloid Metaplasia Non-Hodgkin's Lymphoma (Myelofibrosis)
Prolymphocytic Leukemia Essential Thrombocythemia Polycythemia Vera
Phagocyte Disorders Histiocytic Disorders Chediak-Higashi Syndrome
Familial Erythrophagocytic Chronic Granulomatous Disease
Lymphohistiocytosis Neutrophil Actin Deficiency Hemophagocytosis
Reticular Dysgenesis Histiocytosis-X Langerhans' Cell Histiocytosis
Liposomal Storage Diseases Congenital (Inherited) Immune System
Adrenoleukodystrophy Disorders Alpha Mannosidosis Absence of T and
B Cells SCID Gaucher's Disease Absence of T Cells, Normal B Cell
SCID Hunter's Syndrome (MPS-II) Ataxia-Telangiectasia Hurler's
Syndrome (MPS-IH) Bare Lymphocyte Syndrome Krabbe Disease Common
Variable Immunodeficiency Maroteaux-Lamy Syndrome (MPS-VI) DiGeorge
Syndrome Metachromatic Leukodystrophy Kostmann Syndrome Morquio
Syndrome (MPS-IV) Leukocyte Adhesion Deficiency Mucolipidosis II
(I-cell Disease) Omenn's Syndrome Mucopolysaccharidoses (MPS)
Severe Combined Immunodeficiency (SCID) Niemann-Pick Disease SCID
with Adenosine Deaminase Deficiency Sanfilippo Syndrome (MPS-III)
Wiskott-Aldrich Syndrome Scheie Syndrome (MPS-IS) X-Linked
Lymphoproliferative Disorder Sly Syndrome, Beta-Glucuronidase
Deficiency (MPS-VII) Wolman Disease Inherited Erythrocyte
Abnormalities Other Inherited Disorders Beta Thalassemia Major
Cartilage-Hair Hypoplasia Blackfan-Diamond Anemia Ceroid
Lipofuscinosis Pure Red Cell Aplasia Congenital Erythropoietic
Porphyria Sickle Cell Disease Glanzmann Thrombasthenia Lesch-Nyhan
Syndrome Osteopetrosis Sandhoff Disease Inherited Platelet
Abnormalities Plasma Cell Disorders Amegakaryocytosis/Congenital
Multiple Myeloma Thrombocytopenia Plasma Cell Leukemia
Waldenstrom's Macroglobulinemia Other Malignancies Autoimmune
Diseases Brain Tumors Multiple Sclerosis Ewing Sarcoma Rheumatoid
Arthritis Neuroblastoma Systemic Lupus Erythematosus Ovarian Cancer
Diabetes Mellitus Renal Cell Carcinoma Inflammatory Bowel Diseases
Small-Cell Lung Cancer Testicular Cancer Other Applications Bone
Marrow Transplants Heart Disease (myocardial infarction), either
alone or in combination with enhancing agents such as
erythropoietin Liver Disease Muscular Dystrophy Alzheimer's Disease
Parkinson's Disease Spinal Cord Injury Stroke, either alone or in
combination with enhancing agents such as erythropoietin Peripheral
Vascular Disease Head trauma Ex vivo and In vivo expanded stem and
progenitor cell populations In vitro fertilization application and
enhancement Hematopoietic Rescue Situations (Intense
Chemo/Radiation) Stem cells and progenitor cells derived from
various tissues sources Application in humans and animals Limb
regeneration, alone or in combination with enhancing agents
[0085] Cell therapy is also desirable for treatment of diseases in
which the immune system is sought to be enhanced. One particular
form of cell therapy involves the expansion of T cells that possess
specificity for a distinct antigen, for example a tumor antigen. In
other types of cell therapy, T cells are generated, and
reprogrammed ex vivo for ability to kill a plurality of cells that
express a plurality of markers. Examples of such cell therapy
include expansion of autologous T cells with IL-2, stimulation with
tumor cell lysates, and reintroduction of said cells into the
patient.
[0086] On the other hand, cell therapy may be performed in
situations where suppression of an immune response is desired. In
such situations expansion of cells such as CD4+CD25+ regulatory T
cells is desirable since these cells are capable of inhibiting
immune responses in an antigen-specific manner. Methods for
expansion of these cells are commonly known and include use of
cytokines such as TGF-b.
[0087] One issue in bone marrow homing is that the receptors on
endothelial cells for the glycosylated ligands of circulating cells
are constitutively expressed. These receptors, such as P and E
selectins, induce numerous activities after interacting with cells,
including causing apoptosis or proliferative arrest (Winkler et
al., Blood 2004 103: 1685-1692). Accordingly, the administration of
hematopoietic cells and their subsequent homing to the bone marrow
is dependent on molecules that are constitutively expressed.
[0088] For the purpose of a broader application of this approach
for regenerative medicine in which cells are administered for
non-hematopoietic purposes, the trafficking/homing of cells to the
targeted location is much more complex and involves ligands that
are not constitutively expressed, but expressed as a result of
inflammation or tissue damage. For example, administration of stem
cells for the purpose of treating myocardial infarction depends on
homing of these cells to areas bathed in cytokine locally released
which not only induces expression of E selectin and P selectin on
the endothelium but also mediates chemoattraction to the site. This
tissue localized upregulation of receptors and chemoattractant
agents allows for homing of stem cells into areas of injury.
[0089] Routes of Administration
[0090] Administration of the modified cells is performed in
agreement with standard practices that are known to one skilled in
the art. Several embodiments are possible. For example, routes of
administration may include parenteral, e.g., intravenous,
intradermal, microvascular bed of bone marrow, subcutaneous, oral
(e.g., ingestion or inhalation), trans dermal (topical),
transmucosal, and rectal administration. In certain particular
aspects, the population of modified cells is administered from a
route selected from a group consisting of: intravenously,
intraarterially, intramuscularly, subcutaneously, transdermally,
intratracheally, intraperitoneally, intravitreally, via direct
injection, into bone compartments or into spinal fluid. In some
aspects the cells, compositions or other materials can be used with
a scaffolding support. In certain aspects, the population of
modified cells is administered in or proximal to a site of injury.
In certain aspects, the homing and engraftment takes place within
the bone marrow of the patient in need thereof. In certain aspects,
the cells are administered by multiple routes and/or sites, either
simultaneously or sequentially.
[0091] In another embodiment, the methods, compositions, cells and
other materials can be useful for enhancing functional capabilities
in a wide variety of not only hematological disorders but also
non-hematological disorders. Specifically, the methods,
compositions, cells and other materials may be useful for the
treatment of medical conditions which are amenable to cell therapy.
More specifically, the methods, compositions, cells and other
materials may be useful for the treatment of acute leukemias,
chronic leukemias, myelodysplastic syndromes, stem cell disorders,
myeloproliferative disorders, lymphoproliferative disorders,
phagocyte disorders, histiocytic disorders, lysosomal storage
diseases, congenital immune system disorders, inherited erythrocyte
abnormalities, other inherited disorders, inherited platelet
abnormalities, plasma cell disorder, various malignancies such as
brain tumors or Ewing sarcoma, Autoimmune Diseases, and other
applications such as bone marrow transplants, diabetes, heart
disease, liver disease, hematopoietic rescue situations following
intense chemo/radiation, limb ischemia and limb regeneration
(including cartilage regeneration, skin regeneration, blood vessel
regeneration, etc), cartilage regeneration, skin regeneration,
blood vessel regeneration, etc.
[0092] Furthermore, presented herein is the finding that the
general increased adhesion of cells that have been fucosylated ex
vivo can be utilized for augmented binding to localized niche areas
in absence of chemotactic gradient such as in the context of portal
vein injection or pulmonary artery injection.
[0093] For P-selectin binding cells can be incubated with
anti-CD34.sup.+-PE and with P-selectin isolated from human
platelets. P-selectin binding can be detected with FITC-labeled
S12, a non-blocking mAb to human P-selectin. For E-selectin binding
cells can be incubated with E-selectin/IgM after Fe receptor
blocking. E-selectin can then be detected with FITC-labeled goat
anti-human IgM polyclonal antibodies. Visualization of binding can
be achieved using FACS analysis. Incubation for both P and
E-selectin can be carried out at 4.degree. C. for 20 minutes.
[0094] To confirm a functional consequence of fucosylation
following treatment with fucosyltransferase the cells can be
examined for adhesion to either E-selectin or P-selectin under
physiological shear forces using an in vitro flow chamber rolling
assay system. P-selectin isolated from human platelets can be
immobilized on plates in a parallel-plate flow chamber. A
P-selectin site density of about 145 sites/.mu.m.sup.2 can be used
and measured by binding of .sup.125I-labeled anti-P-selectin mAb
512. For E-selectin soluble human E-selectin can also be
immobilized on plates in a parallel-plate flow chamber at a density
of 200 sites/.mu.m.sup.2, as measured by binding of
.sup.125I-labeled anti-human E-selectin mAb ES1. Sham-treated or
FTVI-treated cells (in Hanks' balanced salt solution and 0.5% human
albumin) can be perfused over P- or E-selectin coated plates at a
wall shear stress of 1 dyn/cm.sup.2. The accumulated number of
rolling cells can be measured with the aid of a videomicroscopy
system coupled to an image analysis system. Specificity of
interaction of cells with the coated plates can be confirmed with
the inclusion of specific inhibitors to the binding and examination
of rolling on plates coated only with human serum albumin.
EXAMPLES
[0095] Examples are provided hereinbelow. However, the presently
disclosed and claimed inventive concept(s) is to be understood to
not be limited in its application to the specific experimentation,
results and laboratory procedures. Rather, the Examples are simply
provided as one of various embodiments and are meant to be
exemplary, not exhaustive.
Example 1
Parameters for Maximal FTVI Activity in Cord Blood
[0096] Enzymatic-mediated fucosylation (.alpha. 1-3-linked fucose
addition to cell-surface glycans) has shown both phenotypic and
functional changes in MNC and CD34.sup.+ cell populations.
[0097] These in vitro studies are structured to examine the various
components integral to the enzymatic-mediated fucosylation using a
cell preparation procedure routinely practiced in the clinic. A
frozen thawed human cord blood mononuclear cell population is
washed by a procedure that involves a 1 to 10 dilution with chilled
10% Dextran-40/5% HSA solution, placing this diluted solution in a
pre-cooled (2-6.degree. C.) centrifuge for 5-10 minutes followed by
mild centrifugation at approximately 550 g for 20 minutes. The
supernatant is discarded while the pellet is resuspended in Hank's
balanced salt solution (HBSS) containing 1% HSA at a target cell
concentration ranging from 0.5.times.10.sup.6 to 1.times.10.sup.9
per ml. This cell population suspended in fucosyltransferase VI
(FTVI) reaction media constitutes the core preparation used to
examine a range of various parameters in order to identify the
optimal conditions for maximal activity of FTVI within this cell
population. The parameters examined are Mn++(over final
concentration ranging from 0.0 to 10 mM), GDP-fucose (over final
concentration ranging from 0.3 to 10 mM), time course analysis with
varying periods of incubation (ranging from 15 minutes to 60
minutes), FTVI (over a 20 fold range of enzyme concentration),
temperature [10.degree. C., 25.degree. C. (considered as room
temperature) and 37.degree. C.] and cell concentration (over final
concentration ranging from 0.5.times.10.sup.6 to 1.times.10.sup.9
per ml). Additional activities within these optimization efforts
include assessing the stability of the fucosylated product with an
examination of HECA-452 binding at 30, 60, 120, 180 and 240 minutes
post termination of reaction and assessing the extent of
fucosylation of additional cell populations. The cell preparation
in reaction mix is incubated for 30 minutes (except for time course
study) with occasional gently mixing. The solutions are then
diluted with cold HBSS 1% HSA, filtered through 70 micron cell
screen and subjected to chilled centrifugation for 12 minutes. The
supernatant is discarded, pellet loosened and resuspended for
either injection or analysis by FACS using a procedure that is
familiar to someone skilled in the art. For FACS analysis, aliquots
of the cells, at approximately 5.times.10.sup.5, are centrifuged at
500.times.g for 13 minutes at 4.degree. C. The supernatant is
discarded, pellet loosened for the addition of flow stain cocktail
containing the appropriate staints) or control cocktail. The
mixture is stored in the dark for 30-40 minutes with occasional
mixing. Each tube is diluted with 3 ml of cold flow wash buffer
followed by centrifugation at 500.times.g for 12 minutes at
4.degree. C. The supernatant is discarded, pellet loosened with the
addition of approximately 200 .mu.l of cold flow buffer or flow fix
buffer. Aliquots of each sample are examined by FACS for percentage
of double positives for CD34.sup.+ vs HECA-452 which is the primary
outcome measure for determining maximal expression of FTVI activity
in the cell mix. Also generated as an outcome measure for
assessment of enzyme activity is the mean fluorescence
intensity
Example 2
Enhanced Engraftment in the Bone Marrow Using a Combination
Approach Consisting of Maximal Fucosylation of Cell Preparation
Plus Exposure to a CD26 Inhibitor
[0098] First, maximal fucosylation of cells in vitro is
accomplished. To accomplish this, washed mononuclear cells (MNCs)
are resuspended in Hank's Balanced Salt Solution (HBSS) at a
concentration of 0.5.times.10.sup.6-1.times.10.sup.9 per ml and
then incubated with a fucosylation mix consisting at final
concentration of 5 mM GDP-fucose, purified human recombinant al-3
fucosyltransferase VI at a predetermined Units per ml (for maximal
fucosylation), and 1-10 mM MnCL.sub.2 in HBSS. This mix is
incubated for 30 minutes at 37.degree. C. or room temperature in a
humidified atmosphere containing 5% CO.sub.2. The period of
incubation could take longer or shorter depending on the incubation
temperature chosen. Following completion of the fucosylation and to
achieve inhibition of CD26 (dipeptidylpeptidase, DPPIV) as part of
the combination approach, this preparation of fucosylated
mononuclear cells (MNCs) is either washed first or directly
incubated with a potent DPPIV inhibitor for 5-15 minutes at room
temperature at a concentration sufficient to achieve complete or
nearly complete inhibition of DPPIV. Following incubation the cell
suspension is volume adjusted with HBSS or another clinically
compatible solution to obtain the appropriate concentration of
cells in preparation for iv injection. As an alternative scenario
for a combination approach, the patients are subjected to systemic
administration of the DPPIV inhibitor at a dose sufficient to
achieve a sustained level in the body for significant inhibition of
bone marrow and circulating plasma DPPIV activity. Subsequent to
systemic pretreatment, MNCs exposed to conditions for maximal
fucosylation are then injected into the patient. The DPPIV
inhibitor can be added simultaneous with the injection of the
fucosylated cell preparation or shortly before. The patients are
prepared for this combined approach by subjecting them to
conditions for myeloablation or even partial myeloablation (mini)
prior to injection of the fucosylated and inhibitor treated cells.
The rate of recovery and extent of chimerism is assessed with an
examination of serially collected blood samples in addition to an
examination of cells obtained from the bone marrow. A multilineage
analysis of the rate of recovery and extent of engraftment and
chimerism is accomplished using cell surface markers specific for
cell types in addition to an examination of mature hematopoietic
cells in the blood stream. These markers and cells are detected
using preparation techniques and FACS analysis procedures that are
familiar to one skilled in the art.
Example 3
Ex Vivo Fucosylation of Mesenchymal Stem Cells
[0099] The introduction of fucose onto surface glycans of
mesenchymal stem cells (MSC) is accomplished by enzymatic transfer
from a donor substrate utilizing an alpha 1-3-fucosyltransferase
(FT). For this transfer to occur the cells at varying
concentrations are exposed to an incubation buffer containing a
number of ingredients each of which has been optimized for
efficient transfer of the fucose, and performed in Hanks balanced
salt solution (HBSS). The substrate, guanosine diphosphate-fucose
(GDP-fucose), at 1 mM is mixed with the FT added at sufficient
activity, in order to achieve maximal transfer of fucose to MSCs.
In addition, MrrCl, at a final concentration of 0-10 mM is added,
as needed, to further accelerate the enzymatic transfer reaction.
The incubation is performed at 37.degree. C. for 40 minutes with
minimum toxicity to the cells.
[0100] Confirmation of fucosylated epitopes on the cells of
interest as means of confirming maximal levels of fucosylation is
verified by Flow Cytometry in order to detect sialyl LewisX (sLeX),
a fucosylation epitope found on both P and E-selectins. The
FT-treated cells are incubated with anti-sLeX mAb HECA 452 (IgM),
followed by treatment with FITC-conjugated fragment to the IgM.
Finally, the sLeX epitopes on the cell surface are visualized using
standard Flow Cytometry procedures.
[0101] To measure P-selectin binding, cells are incubated with
anti-CD34.sup.+-PE and with P-selectin isolated from human
platelets. P-selectin binding is detected with FITC-labeled S12, a
non-blocking mAb to human P-selectin. To measure E-selectin
binding, cells are incubated with E-selectin/IgM after Fe receptor
blocking. E-selectin is then detected with FITC-labeled goat
anti-human IgM polyclonal antibodies. Visualization of binding is
achieved using FACS analysis. Incubation for both P and E-selectin
is carried out at 4.degree. C. for 20 minutes.
[0102] To confirm a functional consequence of fucosylation
following treatment with FT the cells are examined for adhesion to
either E-selectin or P-selectin under physiological shear forces
using an in vitro flow chamber rolling assay system. P-selectin
isolated from human platelets is immobilized on plates in a
parallel-plate flow chamber. A P-selectin site density of about 145
sites/.mu.m.sup.2 is used and measured by binding of
.sup.125I-labeled anti-P-selectin mAb S12. For E-selectin soluble
human E-selectin is also immobilized on plates in a parallel-plate
flow chamber at a density of 200 sites/.mu.m.sup.2 as measured by
binding of .sup.125I-labeled anti-human E-selectin mAb ES1.
Sham-treated or FTVI-treated cells (in Hanks' balanced salt
solution and 0.5% human albumin) is perfused over P- or E-selectin
coated plates at a wall shear stress of 1 dyn/cm.sup.2. The
accumulated number of rolling cells is measured with the aid of a
videomicroscopy system coupled to an image analysis system.
Specificity of interaction of cells with the coated plates is then
confirmed with the inclusion of specific inhibitors to the binding
and examination of rolling on plates coated only with human serum
albumin.
Example 4
Administration of Modified Stem Cells to Bone Marrow Transplant
Patients
[0103] Patients in need of a bone marrow transplant are subjected
to either myeloablative or non-myeloablative conditions. Stem cells
obtained from one of a number of different sources are incubated ex
vivo with fucosyltransferase+GDP-fucose at sufficient
concentrations and for a sufficient period of time to result in
maximal formation of fucosylated product, such as sialyl Lewis X,
on the cell surface. Following treatment, the cell preparation is
washed or directly injected into the patient. Effectiveness of this
application in the patient is determined with accelerated
appearance over time of neutrophils and platelets in the blood
stream compared to patients injected with control untreated stem
cells.
Example 5
Modification of Neural Stem Cells
[0104] A cell population consisting of neural stem cells is treated
with conditions so as to endow increased surface ligands for
enhanced interaction with endothelium. Cells are modified with the
addition of alpha 1-3-linked fucose to cell-surface glycans by ex
vivo treatment of cells with the enzyme alpha 1-2
fucosyltransferase VI. Specifically, cells are treated with 1 mM
GDP fucose, 20 mU/mL {alpha}1-3 fucosyltransferase VI, and 10 mM
MnCl2 in 0.5 mL HBSS containing 1% human serum albumin (HSA) for 30
minutes at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2 under conditions that cause minimum toxicity to CD34.sup.+
cells as tested by propidium iodide staining measured by flow
cytometry. Other modifications of this treatment procedure may be
performed based on the knowledge of one skilled in the art. Said
treated neural stem cells are subsequently assessed for
fucosylation using flow cytometric methodology. The cells are
placed on ice for 5 minutes followed by washing with PBS (1 ml). To
detect the presence of new fucosylated units on cell surface the
cell preparation is treated with the 1.sup.st antibody, anti-CLA
1:200 dilution in blocking buffer (400 .mu.l), and then incubated
for one hour at room temp or overnight at 4.degree.. Cells are then
washed three times with PBS. The secondary antibody
(anti-rat-IgM-PE, 1:200) in blocking buffer (400 .mu.l) is then
added. The preparation is incubated for 2 hrs at room temperature
then rinsed with PBS. The result of this treatment and analysis is
shown in FIG. 1.
Example 6
Modification of Immune Modulatory Cells
[0105] A cell population consisting of cells with immune modulatory
potential is treated with certain conditions so as to endow
increased surface ligands for endothelium. Cells are modified with
alpha 1-3-linked fucose to cell-surface glycans by treatment of
cells with the enzyme alpha 1-2 fucosyltransferase VI.
Specifically, cells are diluted to a concentration of 10(7) per ml
and treated with 1 mM GDP fucose (EMD Bioscienees, San Diego,
Calif.), 20 mU/mL {alpha} 1-3 fucosyltransferase VI (FTVI; EMD
Bioscienees), and 10 mM MnCl.sub.2 in 0.5 mL HBSS containing 1%
human serum albumin (HSA) for 30 minutes at 37.degree. C. in a
humidified atmosphere containing 5% CO.sub.2 under conditions that
cause minimum toxicity to CD34.sup.+ cells as tested by propidium
iodide staining measured by flow cytometry. Other modifications of
this treatment procedure may be performed based on the knowledge of
one skilled in the art. Said treated immune cells are subsequently
assessed for fucosylation status using either a flow cytometrie
methodology (assessment of HECA-453 binding) or functional
methodology (assessment of rolling on endothelium). Cells are
subsequently administered to a patient for immune modulation.
Example 7
Augmenting Efficacy of Tumor Infiltrating Lymphocytes after Ex Vivo
Expansion
[0106] Tumor infiltrating lymphocytes were collected as described
by Zhou et al The Journal of Immunology, 2005, 175: 7046-7052.
Briefly, explants of small (2 mm3) tumor fragments or
1.times.10.sup.6 viable cells of tumor tissue digests were used to
initiate TIL culture in 2 ml of RPMI 1640-based medium (Invitrogen
Life Technologies) containing 10% human serum and 6000 IU/ml IL-2
(Chiron). After 2-4 weeks of culture, several million TIL cells
were usually obtained and screened by IFN secretion assay for
recognition of tumor cells. Antitumor TIL cultures were further
expanded in AIM V medium (Invitrogen Life Technologies)
supplemented with irradiated allogeneic feeder cells, anti-CD3 Ab
(Ortho Biotech), and 6000 IU/ml IL-2. This expansion protocol
typically resulted in 1000-fold expansions of cells by the time of
administration 14-15 days after initiation of the expansions.
Subsequent to expansion cells were harvested, centrifuged, diluted
to a concentration of 10(7) per ml, and treated with 1 mM GDP
fucose (EMD Biosciences, San Diego, Calif.), 20 mU/mL alpha 1-3
fucosyltransferase VI (FTVI; EMD Biosciences), and 10 mM MnCl.sub.2
in 0.5 mL HBSS containing 1% human serum albumin (HSA) for 30
minutes at 37.degree. C. in a humidified atmosphere containing 5%
CO.sub.2 under conditions that cause minimum toxicity to cells, as
tested by propidium iodide staining measured by flow cytometry.
Cells were administered on a weekly basis at a concentration of at
least about 1 million cells, but in some situations up to 100
million cells, over a period of 60-120 minutes. After four cycles
of therapy, tumor regression was noted.
Example 8
Ex Vivo Fucosylation of T Cells
[0107] In order to compare the effects of ex vivo fucosylation on
different T cell types, recombinant FTVI produced in CHO cells was
manufactured at Aragen Bioscience (Morgan Hill, Calif., final
concentration 1100 ug/mL), and FTVII produced in a mouse lymphocyte
line was obtained from Kyowa Hakko Kirin (Japan, final
concentration 150 ug/mL). Frozen human umbilical cord bloods were
purchased from the San Diego Blood Bank (San Diego, Calif.). Unless
otherwise indicated, cells were treated at 10.sup.6 cells/ml for 30
minutes at room temperature with 1 mM GDP .beta.-fucose (EMD
Biosciences, San Diego, Calif.) in Phosphate Buffered Saline (PBS)
containing 1% human serum albumin (HSA, Baxter Healthcare Corp.,
Westlake Village, Calif.) and in the presence of previously
optimized concentrations of FTVI (100 mU/ml) or FTVII (75
.mu.g/ml). The FTVI+GDP-fucose is referred to herein as TZ101
(previously referred to as ASC-101; Targazyme, Inc., Carlsbad,
Calif.), while the FTVII+GDP-fucose composition is referred to
herein as TZ102 (previously referred to as ASC-102; Targazyme,
Inc., Carlsbad, Calif.). Untreated cells were incubated as above,
except that no enzyme was added. Fucosylation levels were
determined by flow cytometry using HECA-452 antibody (BD
Biosciences, San Jose, Calif.), a directly conjugated (FITC), rat
IgM antibody that reacts against a fucosylated (sialyl Lewis X
(sLeX)-modified) form of P-selectin glycoprotein ligand (PSGL)-1
(CD162), also known as cutaneous lymphocyte antigen (CLA). Other
antibodies to CD antigens were also obtained from BD
Biosciences.
[0108] Regulatory T Cells
[0109] Regulatory T cells ("Tregs") were enriched from cord blood
using magnetic bead-isolation (using MACS.RTM. beads, Miltenyi
Biotec Inc., San Diego, Calif.) for CD25.sup.+ cells and expansion
of Tregs with IL-2 and CD3/28 beads, as shown in FIG. 2.
[0110] FIG. 3 demonstrates a flow cytometry analysis of Tregs
expanded in this manner. As shown in the upper right hand panels
(left to right) of FIG. 3, 97.2% of cells are within the lymphocyte
gate, while 98.4% express Treg markers CD25/CD127. As shown in the
lower panels (left to right) of FIG. 3, 98.6% of cells are
CD25.sup.+FoxP3.sup.+; 97.9% are CD4.sup.+FoxP3.sup.+; 98.9% of
cells are CD25.sup.+CD4.sup.+; and 0.52% of cells are
CD25.sup.+CD8.sup.+.
[0111] Tregs were fucosylated with TZ101 (1/25 dilution of TZ101 in
1 mM GDP-Fucose, PBS 1% human serum albumin) for 30 minutes at room
temperature, washed, and assayed for cell surface expression of
sialyl Lewis X (sLeX) using the HECA 452 anti-CLA antibody. The
left side of each dot plot of FIG. 4 shows the isotype control,
while the right side shows the staining with percent CLA positive
cells. As shown in FIG. 4, treatment with TZ101 increased the
percentage of fucosylated Tregs from about 9% to about 63%.
[0112] As shown in FIG. 5, the ability to fucosylate ex vivo
expanded Tregs is unexpected in light of previous studies showing
that TZ101 fucosylated CD34.sup.+ (red curve), CD33.sup.+ (blue
curve), and CD56.sup.+ cells (black curve) at different time points
but did not fucosylate CD3 positive T cells in unexpanded cord
blood (green curve).
[0113] A xenogeneic graft-versus-host (GVHD) model was developed in
which NOD/SCID IL-2R.gamma..sup.null (NSG) mice (Jackson
Laboratory, Bar Harbor, Me.) received sub-lethal whole body
irradiation (300 cGy from a .sup.137Cs source delivered over one
minute by a J. L. Shepherd and Associates Mark I-25 Irradiator, San
Fernando, Calif.) one day prior (Day-1) to intravenous infusion of
human peripheral blood mononuclear cells (PBMC). On Day-0, mice
received PBMC at a dose of 1.times.10.sup.7.
[0114] As shown in FIG. 6, fucosylated Tregs prevented development
of GVHD, while unmanipulated Tregs did not. Sublethally irradiated
NSG mice received Treg or FT-Treg at a cell dose of
1.times.10.sup.6 on Day-1 followed by tail vein injection of PBMC
at cell dose of 1.times.10.sup.7 on Day-0. The mice were followed
for survival and weight. All the Treg recipients were dead at
Day-20; however, the FT-Treg recipients were alive at the last
follow up. While the Treg recipients started losing weight as early
as Day-12, FT-Treg recipients maintained their weight until their
last follow up.
[0115] Cytotoxic T Cells
[0116] Surprisingly, in contrast to Tregs, expanded CD8.sup.+ T
cells were not fucosylated by TZ101 (FTVI+GDP-fucose) but were
fucosylated by TZ102 (FTVII+GDP-fucose).
[0117] In order to generate cytotoxic T cells that were capable of
killing acute myelogenous leukemia (AML) cells, dendritic cells
(DC) were generated from HLA-A*0201 healthy donor monocytes by
adherence and matured and subsequently used as antigen presenting
cells (APC). Healthy donor PBMCs were adhered on 6-well plates at
37.degree. C. in serum-free medium. Cells remaining in suspension
(lymphocytes) from the same donor were removed and pulsed with 40
.mu.g/mL of a peptide (CG1) derived from the myeloid primary
granule protease (PGP) cathepsin G (CG) that has been established
as a myeloid leukemia target. A separate incubation was conducted
with control peptides. The suspension cells were stimulated with
IL-7 (10 ng/mL) and IL-2 (10 ng/mL) for 5 days. Adherent cells from
the initial step were matured into monocyte-derived DC by addition
of GM-CSF (100 ng/mL), IL-4 (50 ng/mL), and TNF-.alpha. (25 ng/mL).
After 5 days, DC were detached and pulsed with appropriate peptides
at 40 .mu.g/mL and subsequently combined with the remainder of
autologous lymphocyte population. Co-cultures were then
re-stimulated with IL-7 (10 ng/mL) and IL-2 (25 ng/mL) for 7 days
to allow for CTL proliferation. On Day-12, cells were harvested and
analyzed by dextramer staining and in vitro cytotoxicity assays to
confirm CTL expansion and specificity. Using this method, CG1-CTL
(experimental effector cells) as well as CTL that target the
HLA-A*0201 HIV Gag (SLYNTVATL) control peptide were generated.
[0118] As shown FIG. 7, TZ102 (FTVII) was capable of fully
fucosylating expanded CTL; however, FTVI did not fucosylate these
cells (data not shown).
[0119] In order to compare the efficacy of FTVII-treated CTL to
unmanipulated CTL, a xenogeneic AML model was developed. Cells from
the human U937 leukemia cell line transduced with HLA-A2 and GFP
were administered to NSG mice on Day-0. Cytotoxic T cells were
expanded against CG1 as described above. Fucosylated or
non-fucosylated T cells were administered to NSG mice with U937
leukemia on Day-1, and bone marrow was harvested 14 days later and
assessed for the presence of U937 cells (upper left quadrant of
FIG. 8). There were significantly fewer leukemia cells in mice
receiving fucosylated T cells (arrows in FIG. 8).
[0120] One of ordinary skill in the art will appreciate that these
methods, compositions, and cells are and may be adapted to carry
out the objectives and obtain the ends and advantages mentioned, as
well as those inherent therein. The methods, procedures, and
devices described herein are presently representative of particular
embodiments and are exemplary and are not intended as limitations
on the scope of the technology. Changes therein (including, but not
limited to, changes in method steps as well as sequence of method
steps) and other uses will occur to those of ordinary skill in the
art which are encompassed within the spirit of the technology and
are defined by the scope of the disclosure. It will be apparent to
one of ordinary skill in the art that varying substitutions and
modifications can be used or substituted into any of the
embodiments described herein may be made without departing from the
scope and spirit of the described technology. Examples of such
substitutions are non-natural enzymes and sugars. Those of ordinary
skill in the art recognize that the aspects and embodiments set
forth herein may be practiced separate from each other or in
conjunction with each other. Therefore, combinations of separate
embodiments are within the scope of the technology as disclosed
herein.
* * * * *