Note: Descriptions are shown in the official language in which they were submitted.
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
1
METHODS AND COMPOSITIONS FOR BINDING
HEMATOPOIETIC STEM CELLS
TECHNICAL FIELD
The present invention relates generally to methods and compositions for
5 binding hematopoietic stem cells. The invention is more particularly
directed to
methods for immobilizing, purifying or labeling stem cells. The invention is
also
directed to methods for targeting agents, such as therapeutic agents, to stem
cells.
BACKGROUND OF THE INVENTION
Gene therapy represents a promising approach to the prevention and
10 treatment of a variety of diseases. In practice, however, the application
of genetic
therapies has been limited by practical problems, such as the inability to
target an agent
specifically to a desired cell type or the limited lifespan of a genetically
altered cell.
The use of multipotent hematopoietic progenitor cells as vehicles for
gene therapy could potentially overcome such difficulties. These stem cells,
which are
15 characterized by a cell surface marker known as CD34, could be genetically
modified in
vitro, and then transplanted into a patient using routine techniques.
Following
transplantation, the transgenic cells can home directly to bone narrow and, if
required,
fully repopulate and eventually reconstitute all of the blood cells of the
host. In this
fashion, the desired gene product may be produced in a constitutively
regenerating
20 population of cells in vivo.
Previous attempts to take advantage of these properties of hematopoietic
stem cells, however, have been hampered by difficulties associated with in
vitro
manipulation. In culture, these progenitor cells lose their pluripotency and
differentiate.
To avoid this problem, stem cells have been co-cultured with microvascular
endothelial
25 feeder cells (primary cells from the brain of non-human sources) and
certain cytokines.
Under such conditions, an expanded population of stem cells has been shown to
remain
CD34+ and CD38- while interacting with feeder cells (see Davis et al., Blood
85:1751-
61, 1995). Adhesion to these endothelial cells appears to be a critical
requirement for
maintaining the pluripotent CD34 phenotype. Such co-culture techniques appear
to
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
7
stabilize the dedifferentiated state of the stem cells sufficiently to allow
genetic
manipulation. Unfortunately, serious drawbacks (such as the limited lifespan
of the
feeder cells, potential biohazards and cross-species immune responses) prevent
serious
consideration of this method for therapeutic use. New methods for binding
5 hematopoietic stem cells are needed that avoid these drawbacks.
Agents that bind stem cells would also aid in purification of such cells
prior to, for example, bone marrow transplantation. Current affinity
procedures often
involve recognition of an epitope of the CD34 antigen. Within such procedures,
problems with specificity and release from the affinity matrix have been
encountered.
10 Accordingly, there is a need in the art for improved methods for
immobilizing and purifying hematopoietic stem cells that overcome the
disadvantages
encountered with existing techniques. The present invention fulfills this need
and
provides further related advantages.
SUMMARY OF THE INVENTION
15 Briefly stated, this invention provides methods and compositions for
binding hematopoietic stem cells. Within one aspect, the present invention
provides
methods for immobilizing hematopoietic stem cells, comprising contacting a
biological
sample containing hematopoietic stem cells with a binding partner, wherein the
binding
partner binds to a sialylated carbohydrate chain comprising the structure
NeuAca2-
20 3Gal~l-4. Within specific embodiments, the binding partner may be attached
to a solid
support before or after complex formation. Suitable binding partners include
antibodies
and antigen-binding fragments thereof and lectins. Within further preferred
embodiments, the sialylated carbohydrate chain comprises the structure NeuAca2-
3(Gal~il-4GlcNAc~i1-3)", wherein n is an integer ranging from 1 to 100, and
more
25 preferably from 2 to 50.
Within a related aspect, methods for purifying hematopoietic stem cells
are provided. Such methods comprise: (a) contacting a biological sample
containing
hematopoietic stem cells with a binding partner that binds to a sialylated
carbohydrate
chain comprising the structure NeuAca2-3Ga1(31-4 to form a stem cell-binding
partner
30 complex: and (b) separating the stem cell-binding partner complex from
unbound
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
3
biological sample. To facilitate separation, the binding partner may be
attached to a
solid support before or after complex formation. Suitable binding partners
include
antibodies and antigen-binding fragments thereof and lectins. Within further
preferred
embodiments, the sialylated carbohydrate comprises the structure NeuAcoc2-
3(Gal~il-
5 4GlcNAc~i1-3)", wherein n is an integer ranging from 1 to 100, and more
preferably
from 2 to 50.
Within further aspects, the present invention provides methods for
targeting an agent to a hematopoietic stem cell, comprising contacting a
biological
sample containing hematopoietic stem cells with a binding partner that binds
to a
sialylated carbohydrate chain comprising the structure NeuAcoc2-3Gal~il-4,
wherein the
binding partner is associated with an agent. Suitable binding partners include
antibodies and antigen-binding fragments thereof and lectins. Within further
preferred
embodiments, the sialylated carbohydrate chain comprises the structure NeuAca2-
3(Gal~il-4GlcNAc(31-3)", wherein n is an integer ranging from 1 to 100, and
more
15 preferably from 2 to 50. Suitable agents include toxic compounds,
therapeutic
compounds and detectable markers.
The present invention also provides kits for use in the above methods.
One such kit, for use in immobilizing hematopoietic stem cells, comprises: (a)
a
binding partner for stem cells, wherein the binding partner binds to a
sialylated
carbohydrate chain comprising the structure: NeuAcoc2-3Ga1(31-4; and (b) a
wash
solution, wherein the binding partner and wash solution are provided in
separate
compartments or containers. The binding partner may, but need not, be attached
to a
support such as a tissue culture dish. Within another embodiment a kit for use
in
separation andJor purification of stem cells is provided, comprising: (a) a
binding
partner for stem cells, wherein the binding partner binds to a sialylated
carbohydrate
chain comprising the structure: NeuAcoc2-3Gal~i1-4; and (b) a solid support.
In a
further embodiment, the present invention provides kits for use in labeling or
sorting
hematopoietic stem cells, comprising (a) a binding partner for stem cells
associated with
a detectable marker, wherein the binding partner binds to a sialylated
carbohydrate
chain comprising the structure: NeuAcoc2-3Gal~ 1-4; and (b) a detection
reagent,
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063 -
4
wherein the binding partner and detection reagent are provided in separate
compartments or containers.
Within further embodiments, the present invention provides
pharmaceutical compositions, comprising: (a) an agent associated with a
binding
5 partner for stem cells, wherein the binding partner binds to a sialylated
carbohydrate
chain comprising the structure: NeuAca2-3Ga1~1-4; and (b) a pharmaceutically
acceptable carrier.
The compositions of the present invention (and agents therein) are also
provided for use as a medicament, and for use for the manufacture of a
medicament for
10 the treatment of a disease, e.g., by gene therapy.
These and other aspects of the present invention will become apparent
upon reference to the following detailed description and attached drawings.
All
references disclosed herein are hereby incorporated by reference in their
entirety as if
each was incorporated individually.
15 BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a graph illustrating the binding of monoclonal antibody
NUH2 to neoglycoproteins within an ELISA. Four different glycoproteins were
used:
"O" 3'SL-APEA-BSA; "O" 3'SL-APD-BSA; "O" 3'SL-BSA; and " X " M6-BSA.
Three comprise 3'sialyllactose (3'SL) linked to bovine serum albumin (BSA),
either
20 directly or via 2-(4-aminophenyl)ethylamine (APEA) or aminophenylene
diamine
(APD). The fourth is M6 linked directly to BSA. The results are presented as
OD4sa as
a function of neoglycoprotein concentration.
Figure 2 is a histogram illustrating the immobilization of hematopoietic
stem cells using representative binding partners. The results are presented as
25 fluorescence units for each binding partner, as indicated.
Figure 3 is a histogram illustrating the immobilization of hematopoietic
stem cells using representative binding partners. The results are presented as
fluorescence units for each binding partner, as indicated.
Figure 4 presents the results of two-color FACS analysis comparing the
30 binding of the representative binding partner NUH2 to surface markers
present on
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
human cord blood cells to the binding observed for the anti-CD34 antibody
HPCA2-PE.
The data are presented as contour plots which represent the bivariate
distribution of
green and red fluorescence. Contour lines known as isopleths are drawn to
express a
cell count at a particular set number.
5 Figure 5 presents the results of two-color FACS analysis comparing the
binding of the control IgM antibody to surface markers present on human cord
blood
cells to the binding observed for the anti-CD34 antibody HPCA2-PE. The data
are
presented as contour plots which represent the bivariate distribution of green
and red
fluorescence. Contour lines known as isopleths are drawn to express a cell
count at a
particular set number.
DETAILED DESCRIPTION OF THE INVENTION
As noted above, the present invention is directed to methods and
compositions for binding hematopoietic stem cells. The methods described
herein
employ a binding partner that binds to a sialylated lactosamine structure on
the surface
I S of such stem cells to form a binding partner-stem cell complex. The
formation of such
complexes facilitates a variety of manipulations, such as, for example,
immobilization,
purification, identification and targeting of hematopoietic stem cells.
Compositions
according to the present invention generally comprise a binding partner, which
may be
free, attached to a support material or linked to a label or therapeutic
agent, depending
20 on the intended use.
Prior to setting forth the invention in detail, it may be helpful to an
understanding thereof to provide definitions of certain terms to be used
herein. As used
herein, the term "binding" refers to a noncovalent association between two
separate
molecules (each of which may be free (i.e., in solution) or present on the
surface of a
25 cell or a solid support), such that a "complex" is formed. Such a complex
may be free
or immobilized (either covalently or noncovalently) on a support material. The
ability
of one molecule to bind to another molecule may generally be evaluated by
determining
a binding constant for the formation of the complex. The binding constant is
the value
obtained when the concentration of the complex is divided by the product of
the
30 component concentrations. In general, two compounds are said to "bind" in
the context
CA 02304961 2000-03-27
WO 99/15628 PCT/IJS98/20063 -
6
of the present invention when the binding constant for complex formation
exceeds
about 102 L/mol.
A binding constant may be determined using methods well known to
those of ordinary skill in the art. For example, the binding constant for the
formation of
5 a complex between a relatively small carbohydrate and a macromolecule
binding
partner may be determined using equilibrium dialysis. Briefly, two chambers of
known
volume are separated by a dialysis membrane that allows transfer of the small
molecular
weight carbohydrate, but not the macromolecule binding partner (e.g., an
antibody).
The carbohydrate is labeled with a reporter group, such as tritium, and is
added to the
10 solution in chamber 1, while the binding partner is placed in the solution
in chamber 2.
The carbohydrate molecules are then allowed to diffuse into chamber 2 until
transfer of
the carbohydrate reaches equilibrium (generally about 1 to 3 days). The
binding
constant may then be determined by measuring the amount of carbohydrate in
each
chamber (which may be readily determined using the reporter group) and using
equation
15 I:
K - fcBl
(~~Bl
20
I
where [C] is the concentration of uncomplexed carbohydrate, [B] is the
concentration of
uncomplexed binding partner and [CB] is the concentration of complex. Since
[C] is
the concentration of carbohydrate in chamber 1 (i.e., [C1]); [B] is difference
between
the original concentration of binding partner (i.e., [Bo]) and [CB]; and [CB]
is the
25 difference between the concentration of carbohydrate in chamber 2 and the
concentration of carbohydrate in chamber 1 (i.e., [C2]-[C1]), equation I may
be
rewritten in measurable terms as equation II:
E~2 )-(~~ l
f~~ ~fB; )-(L~21-U1 J»
30
11
CA 02304961 2000-03-27
WO 99/15628 PCT1US98/20063 -
7
It has been found, within the context of the present invention, that
sialylated lactosamine structures are expressed selectively on hematopoietic
stem cells.
In other words, sialylated lactosamine structures are present on the surface
of a
hematopoietic stem cell at higher concentrations, or in a conformation that
favors
binding to a binding partner, relative to T cells. It has also been found that
binding
partners to these sialylated type 2 chains can be used to select and
immobilize
hematopoietic stem cells. Accordingly, a "binding partner," in the context of
this
invention, is any agent, such as a compound or a cell, that binds to a
sialylated
carbohydrate chain comprising the structure NeuAcoc2-3Gal~i1-4R and/or to a
sialylated
linear or branched lactosamine comprising the structure NeuAca2-3(Ga1~31-
4GlcNAc(31-3)"-R, wherein n is an integer ranging from 1 to 100, preferably
from 1 to
50, and more preferably from 2 to 50, and wherein R represents the remainder
of the
molecule, if any. For example, R may be a saccharide or non-saccharide moiety,
such
15 as a protein or lipid. Such moieties may be included, for example, to aid
in purification,
detection or immobilization of the carbohydrate. Representative carbohydrate
structures
in which R is a lipid include:
and:
NeuAca2-3Galø 1-4GlcNAc(i 1-3Gal(i 1-4G~NAca 1-3Ga1(31-4Gb(31- lCeranvde
NeuAca2-3Gal~ 1-4GIcNAc(31\
6
Gal~i 1-4GlcNAc~ 1-3Gal~i 1-4Glcp 1- lCeranide
/3
NeuAca2-3Gal(i 1-4G~NAc(31
Representative structures in which R is a protein are:
NeuAca2-3Gal~i 1-4GlcNAc~i 1-2Manoc 1~
6
Mardi 1-4GlcNAc~i 1-4GlcNAc(31-Aso-protem
/3
NeuAca2-3Gal~i 1-4GxNAc~i 1-2Mana 1
and:
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
8
NeuAca2-3Ga1(3 I-4GkNAc(3 I-3 Cral(i 1-3G~a~lAca I-Ser-prote~
Any agent (e.g., a cell or a molecule) that satisfies the above
5 requirements may be a binding partner. Within one preferred embodiment, the
binding
partner comprises an antibody, or a binding fragment thereof, raised against a
suitable
immunogen. Suitable immunogens include gangliosides adsorbed and coated on
acid-
treated Salmonella minnesotae, human fetal erythrocytes from cord blood or
human
colonic adenocarcinoma cells.
10 Antibodies may be prepared by any of a variety of techniques known to
those of ordinary skill in the art. See, e.g., Harlow and Lane, Antibodies: A
Laboratory
Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an
immunogen
is initially injected into any of a wide variety of mammals (e.g., mice, rats,
rabbits,
sheep or goats). Following one or more injections, the animals are bled
periodically.
15 Polyclonal antibodies specific for the immunogen may then be purified from
such
antisera by, for example, affinity chromatography using the immunogen coupled
to a
suitable solid support.
Monoclonal antibodies specific for the carbohydrate structure of interest
may be prepared, for example, using the technique of Kohler and Milstein, Eur.
J.
20 Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods
involve
the preparation of immortal cell lines capable of producing antibodies having
the
desired specificity from spleen cells obtained from an animal immunized as
described
above. The spleen cells are immortalized by, for example, fusion with a
myeloma cell
fusion partner, preferably one that is syngeneic with the immunized animal.
Single
25 colonies are selected and their culture supernatants tested for binding
activity against a
sialylated carbohydrate as described herein. Hybridomas secreting antibodies
having
high reactivity and specificity are preferred.
Monoclonal antibodies may be isolated from the supernatants of growing
hybridoma colonies, with or without the use of various techniques known in the
art to
30 enhance the yield. Contaminants may be removed from the antibodies by
conventional
techniques, such as chromatography, gel filtration, precipitation, and
extraction.
Antibodies having the desired activity may generally be identified based on
their ability
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
9
to bind to one or more appropriate neoglycoproteins in an immunoassay.
Suitable
neoglycoproteins include those containing one of the following structures:
NeuAca2-3Ga1(31-4(Glc)-APD-HSA; or
NeuAca2-3(Gal(31-4GIcNAc(31-3)"Gal(31-4(Glc)-APD-HSA where n is defined
above; or
NeuAca2-3Ga1(31-4GlcNAc(31 ~
6
Ga1~31-4GlcNAc(31-3Ga1(31-4(G~}-APD-HSA
NeuAca2-3Gal(31-4GlcNAc(31
The above glycoproteins are conjugates of carbohydrate structures to human or
bovine
serum albumin (HSA or BSA, respectively). Since albumin is non-glycosylated,
the
10 resulting neoglycoprotein contains only the carbohydrate structures
chemically linked to
the molecule. The first neoglycoprotein recited above is commercially
available from
several sources and both molecules can be synthesized by those skilled in the
art.
Typically, the carbohydrate is linked by the spacer acetylphenylenediamine
(APD) by
reductive amination of the reducing end of the carbohydrate chain. In these
cases, the
15 glucose is reduced, the ring opens and creates an aminoalditol. The glucose
unit is
therefore displayed in parenthesis.
An antibody may be further characterized by subjecting human blood
from the umbilical cord to two color fluorescence activated cell sorter
analysis (FACS
analysis). One antibody may directed against the hematopoietic stem cell
marker CD34.
20 Such an antibody is commercially available labeled with the red fluorescent
marker,
phycoerythrin from Becton Dickinson, San Jose, CA (HPCA-2PE). The test
antibody
may be labeled with a green fluorescent marker (fluorescein) either directly
(using, for
example, marker obtained from Molecular Probes, Inc., Eugene, OR) or
indirectly (e.g.,
using fluorescein-labeled goat anti-mouse IgM, available from KPL,
Gaithersburg,
25 MD). Antibodies that result in the labeling of all red fluorescent cells
with green
fluorescence are preferred.
One suitable binding partner is the antibody NUH2 (see U.S. Patent No.
5,227,160). This antibody is an IgM and was developed from a hybridoma
produced
from mice immunized with disialogangliosides from human colonic adenocarcinoma
CA 02304961 2000-03-27
WO 99/15628 PCTNS98/20063
10
cells coated on acid-treated, Salmonella minnesotae (see Nudelman, J. Biol.
Chem.
2b4:18719-25, 1989). Antibody NUH2 has been found to bind the two
neoglycoproteins recited above and also to branched disialogangliosides
containing
sialylated lactosamine structures. In two color FACS analysis, antibody NUH2
also
5 labels all CD34+ cells identified by the commercially-available antibody
HPCA-2-PE.
Within another preferred embodiment, the binding partner comprises a
lectin, or a derivative such as an altered or truncated form that retains
binding
properties, and combinations of lectins and/or derivatives. One suitable
truncated form
is a complementarity-determining region (CDR) domain (see Saragovic et al.,
Science
10 253:792-95, 1991). Suitable lectin binding partners include, but are not
limited to, plant
lectins such as Tomato lectin, Maackia amurensis lectin, Sambucus nigra
lectin,
Triticum vulgaris lectin and Erytheina cristagalli lectin; mammalian lectins
such as
sialoadhesins (see Kelm et al., Glycoconjugate J. 13:913-26, 1996) and
galectins; and
bacterial lectins such as the carbohydrate receptors of Helicobacter pylori
(see Teneberg
15 et al., J. Biol. Chem. 272:19067-71, 1997).
Binding partners may generally be identified by evaluating the ability of
a putative binding partner to bind a test sialylated carbohydrate chain
comprising the
NeuAca2-3Gal~i1-4R structure, as described above. Any of a number of well
known
binding assays may be employed for screening putative binding partners,
including
20 immunoassays. Binding constants for the interaction between binding partner
and test
carbohydrate may be measured as described above.
Test sialylated carbohydrate chains may generally be prepared from
commercially available materials, using methods that will be apparent to those
of
ordinary skill in the art. For example, lactosamine chains of varying lengths
may
25 generally be prepared according to the method of Srivastava et al., J.
Carbohydrate
Cl:em. 10:927-933 (1991). Such chains may be sialylated using
sialyltransferases or
transsialidase enzymes. Sialyltransferases enzymatically transfer sialic acid
(NeuAc)
from the sugar nucleotide CMP-NeuAc to the acceptor carbohydrate chain.
Transialidases transfer sialic acid between sialylated and non-sialylated
carbohydrate
30 chains. One example is the transfer of NeuAc from 3'sialylactose to lacto-N-
neotetraose
to produce 3'sialylacto-N-neotetraose as depicted in the neoglycoprotein
above.
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
11
A "biological sample," as used herein, refers to any tissue sample or
preparation of cells that contains hematopoietic stem cells, including blood,
bone
marrow, huffy coat cells, cord blood and cells that are grown and/or expanded
in vitro.
Such preparations may, but need not, be treated and/or fractionated prior to
use within
5 the methods of the present invention. For example, a biological sample may
be a blood
preparation, with or without cytokine treatment.
As discussed in further detail below, a binding partner can be used to
immobilize stem cells. The term "immobilization" is used herein to refer to a
noncovalent interaction between a stem cell and a binding partner, resulting
in the
10 formation of a stem cell-binding partner complex, wherein the binding
partner or the
complex is attached to a solid support. In one preferred embodiment, the
binding
partner binds to the stem cell prior to immobilization of the complex. Within
another
preferred embodiment, the binding partner is attached to the solid support
before contact
with the stem cells. The term "attach" or "attachment," as used herein, refers
to a
15 covalent or noncovalent interaction between a binding partner or stem cell-
binding
partner complex and a solid support, such that the binding constant of the
interaction is
at least 103 IJmol.
Any of a variety of techniques known to those in the art for attachment to
a solid support, and amply described in the patent and scientific literature,
may be
20 employed. Attachment may generally be achieved through noncovalent
association,
such as adsorption or affinity, or via covalent attachment. Attachment of a
binding
partner or complex by adsorption may be achieved by contact, in a suitable
buffer, with
a solid support for a suitable amount of time. The contact time varies with
temperature,
but is generally between about 5 seconds and 1 day, and typically between
about 10
25 seconds and 2 hours. Attachment by affinity is generally achieved using a
support that
contains a compound that binds to the binding partner and/or complex (e.g., a
support to
which avidin is attached may be used to attach a binding partner associated
with biotin).
Such a compound may itself be attached by adsorption or covalently.
Covalent attachment of a compound to a solid support may be a direct
30 linkage between a binding partner or complex and functional groups on the
support, or
may be a linkage by way of a cross-linking agent. Attachment using a cross-
linking
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
12
agent may generally be achieved by first reacting the support with a
bifunctional reagent
that will react with both the support and a functional group, such as a
hydroxyl or amino
group, on the compound. For example, a binding partner may be bound to a
support
having an appropriate polymer coating using benzoquinone, by condensation of
an
5 aldehyde group on the support with an amine and an active hydrogen on the
binding
partner (see, e.g., Pierce Immunotechnology Catalog and Handbook (1991) at A12-
A13)
or by condensation of an amino group on the support with a carboxylic acid on
the
binding partner.
Within some embodiments of the present invention, as discussed below,
10 an agent may be associated with a binding partner such that the agent can
be targeted to
hematopoietic stem cells. An "agent" may be a compound or a cell, and may be a
therapeutic compound, a cytotoxic compound or a label or detectable marker
suitable
for labeling hematopoietic stem cells {e.g., for cell sorting or in vivo
imaging). Such
association may be noncovalent (such as by way of hydrophobic interactions or
15 incorporation into a liposome), but is preferably covalent and may be
achieved by
standard recombinant or chemical means. "Targeting," within the present
invention,
refers to specifically directing an agent to hematopoietic stem cells in vitro
or in vivo,
such that the agent is delivered to hematopoietic stem cells more efficiently
than to
other cell types. For example, a binding agent associated with a detectable
marker may
20 be used to identify hematopoietic stem cells within a mixture of cells
using standard
techniques (e.g., FACS). For successful bone marrow transplantation, however,
the
population of hematopoietic stem cells need only be enriched several fold and
depleted
of T cells and other contaminating cells that promote graft vs. host disease.
Within one aspect of the present invention, methods are provided for
25 immobilizing hematopoietic stem cells. A biological sample containing such
stem cells
is initially contacted with a binding partner. The binding partner may be
attached to a
solid support, or such attachment can be achieved after complex formation.
Preferred
solid supports for expansion and gene transduction include tissue culture
dishes, hollow
fiber bioreactors and bags, such as blood- and cell-collection bags. For
purification
30 purposes, chromatography matrices are preferred supports. In general, an
excess of
CA 02304961 2000-03-27
WO 99/15628 PCTN598I20063
13
binding partner over the number of stem cells is employed, to facilitate the
formation of
stem cell-binding partner complexes.
Suitable conditions for contacting the binding partner with the biological
sample are generally conditions that favor complex formation and maintain cell
5 viability. Such conditions may be readily determined by one of ordinary
skill in the art
by evaluating the binding constant for complex formation at a series of
different
conditions. Suitable conditions include physiological conditions, such as pH
7.4
isotonic saline (e.g., O.15M NaCI) at room temperature. It will be evident to
those of
ordinary skill in the art that the amount of binding partner necessary to
achieve adequate
complexation of the stem cells in a given biological sample will depend upon
the
concentration of stem cells within the sample, the binding constant for the
given binding
partner and the other materials present in the biological sample. In general a
concentration of binding partner ranging from about 0.1 p,g/mL to 10 mg/mL,
and
typically from about 1 pg/mL to 1 mg/mL, is sufficient.
15 After complex formation, the stem cell-binding partner complex may be
attached to a solid support if necessary. The remainder of the biological
sample may
then be removed from the immobilized stem cells by any of a variety of known
techniques such as, for example, filtration and/or washing with an appropriate
buffer.
Hematopoietic stem cells immobilized as described herein may generally
20 be used for a variety of in vitro manipulations, including expansion and
gene
transduction. The present immobilization procedure generally has the advantage
of
permitting expansion of stem cells in the dedifferentiated state.
Within a related aspect, the present invention provides methods for
purifying hematopoietic stem cells. "Purification" refers to a separation of
the stem
25 cells from at least a portion of the components of the biological sample,
and purified
stem cells may be present within a stem cell-binding partner complex. Within
this
aspect, a biological sample is contacted with a binding partner as described
above. If
the binding partner was not attached to a support prior to contact with the
biological
sample, the complex is attached after such contact and separated from the
unbound
30 portion of the biological sample. Cells may then be released from the
binding partner
using any suitable technique. Preferably, release is achieved by incubation
with high
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
14
concentrations of carbohydrate hapten to compete for binding to the binding
partner.
Alternatively, cells may be eluted by incubation under conditions that
diminish or
eliminate the activity of the binding partner (e.g., a change in pH and/or
cation
concentration). Under appropriate circumstances, cells may be eluted from
5 immobilized binding partners by mechanical agitation. Optionally, further
purification
steps may be performed using other affinity materials.
In other aspects, a binding partner as described herein may be used to
target an agent to hematopoietic stem cells in vitro or in vivo. As noted
above, any of a
variety of agents associated with a binding partner may be directed
specifically to
10 hematopoietic stem cells. For example, an agent may be a detectable marker
suitable
for cell identification and sorting in vitro or imaging in vivo. Such markers
are well
known in the art and include radionuclides, luminescent groups, fluorescent
groups,
enzymes (such as horseradish peroxidase), substrates, cofactors, inhibitors,
dyes,
constant immunoglobulin domains and biotin. Alternatively, an agent may be a
15 therapeutic or cytotoxic compound. Other agents include polynucieotides
encoding a
detectable marker, therapeutic agent or cytotoxic compound.
To deliver an agent in vitro or ex vivo, a biological sample is contacted,
as described above, with the agent associated with the binding partner.
Unbound
binding partner is then removed by standard techniques, such as filtration,
20 centrifugation, precipitation, elution or other appropriate means. For
detectable
markers, the agent may then be detected by any appropriate method known to
those of
ordinary skill in the art, the selection of which will depend in part upon the
agent used.
For example, a fluorescent agent, such as fluorescein, associated with a
binding partner
may be used for FACS analyses, using well known techniques.
25 For in vivo use, an agent associated with a binding partner is typically
administered to a subject in the form of a pharmaceutical composition. A
pharmaceutical composition may be a sterile aqueous or non-aqueous suspension
or
emulsion, which additionally comprises a pharmaceutically acceptable carrier
(i.e., a
non-toxic material that does not interfere with the activity of the active
ingredient). Any
30 suitable carrier known to those of ordinary skill in the art may be
employed in the
pharmaceutical compositions of the present invention. Representative Garners
include
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
15
physiological saline solutions, gelatin, water, alcohols, natural or synthetic
oils,
saccharide solutions, glycols, injectable organic esters such as ethyl oleate
or a
combination of such materials. Optionally, a pharmaceutical composition may
additionally contain preservatives and/or other additives such as, for
example,
5 antimicrobial agents, anti-oxidants, chelating agents and/or inert gases,
and/or other
active ingredients. For imaging uses, after passage of sufficient time to
allow
localization of the detectable marker, the binding partner binds to the
hematopoietic
stem cells and can be detected using conventional imaging techniques, such as
x-ray
technologies.
10 Within further aspects of the present invention, kits for use in any of the
methods described herein are provided. Kits for use in immobilizing
hematopoietic
stem cells generally contain a binding partner, which may be attached to a
tissue culture
dish. For separation and purification of stem cells, a kit may contain a
binding partner
in combination with a filter or chromatographic support. Kits for use in
labeling, cell
15 sorting or in vivo imaging typically contain a binding partner associated
with a
detectable marker. For therapeutic use, a kit may contain binding partner
associated
with a therapeutic agent. In addition to the above components, one or more
additional
compartments or containers of a kit generally enclose elements, such as
reagents,
buffers and/or wash solutions, to be used in the method.
20 The following Examples are offered by way of illustration and not by
way of limitation.
CA 02304961 2000-03-27
WO 99/15628 PCTNS98/20063
16
EXAMPLES
Example 1
IDENTIFICATION OF SIALYLATED LACTOSAMINE STRUCTURES
5 ON HEMATOPOIETIC STEM CELLS
This Example demonstrates use of antibody specific for CD34+ cells to
identify sialylated lactosamine structures on the surface of hematopoietic
stem cells.
Monoclonal antibody NUH2 (ATCC Accession Number HB 9762) was
employed within enzyme-linked immunoassays (ELISAs) for binding to
carbohydrate
10 structures. Monoclonal antibody NUH2 is an IgM isotype that was developed
by
immunizing mice with disialogangliosides isolated from colon adenocarcinoma
and
adsorbed on acid-treated Salmonella minnesotae (see U.S. Patent No.
5,227,160).
To generate a neoglycoprotein library, purified oligosaccharides were
chemically coupled to human serum albumin. Each neoglycoprotein molecule
15 contained about 10 to 15 chains of one specific purified oligosaccharide.
For screening,
the neoglycoprotein library was coated onto the surface of the wells in a
microtiter plate
by incubating the wells filled (100~t1) with neoglycoproteins diluted in
phosphate-
buffered saline, pH 7.4 (PBS) overnight at 4°C. Wells were then blocked
by adding 2%
bovine serum albumin in PBS and incubating for 2 hours at room temperature.
After
20 washing the wells with PBS, monoclonal antibody diluted in PBS at lOp,g/ml
(100w1)
was added to all of the wells. The microtiter plate was incubated at room
temperature
for 2 hours, after which the plate was washed and the wells were filled
(100p,1) with
peroxidase-labeled goat anti-mouse IgM (KPL labs, Gaithersburg, MD) at a
concentration of 1 p,g/ml and incubated for 1 hour at room temperature. The
plate was
25 then washed and 100 ~tl/well of TMB reagent (KPL labs, Gaithersburg, MD)
was added.
After 5 minutes, 1001 of 1M phosphoric acid was added to each well to stop the
color
reaction. The intensity of color in each well was then determined by
determining the
absorbance of light at 450 nm using the Titertek Multiskan MCC/340 (Flow
Laboratories, Inc., McLean, VA).
30 As shown in Figure 1, monoclonal antibody NUH2 binds 3'sialyllactose-
containing structures.
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
17
Example 2
IMMOBILIZATION OF STEM CELLS USING A BINDING PARTNER
This Example illustrates the use of an antibody binding partner to
5 immobilize hematopoietic stem cells.
Immobilization of CD34+ cells to surfaces coated with antibodies and
lectins was tested by a static cell adhesion assay. Antibodies and lectins
diluted in a
buffer containing IOmM Tris, O.15M NaCI, 1mM CaCl2 at pH 7.4 were added to
wells
of a 96 well microtiter plate and incubated overnight at 4°C. Bovine
serum albumin
10 (2% in TrisCa buffer) was added to each well and incubated at room
temperature for at
least 2 hours in order to reduce nonspecific binding. CD34+ cells (10~
cells/ml),
purchased from Poetic Technologies Inc. (Gaithersburg, MD) were incubated with
calcein AN (Molecular Probes, Eugene OR) diluted in Dulbecco's PBS (DPBS) at a
concentration of 2p,g/ml for 20 minutes at 37°C. After washing the
microtiter plate 5
15 times with DPBS, 100 ~1 of cells were added to each well. The plate was
incubated in a
stationary position for 25 minutes at room temperature. After incubation, the
plate was
inverted in a wash chamber (GlycoTech Corp., Rockville MD), for exactly 6
minutes.
The plate was then removed from the chamber, and fluorescence of each well was
determined at excitation and emission wavelengths of 485 and 530, respectively
20 (Cytofluor 2350, PerSeptive Biosystems).
The results are presented in Figures 2 and 3. CD34+ cells bound to the
attached antibody NUH2 and to the lectins Maackia amurensis lectin, Tomato
lectin and
sialoadhesin, but not to negative control proteins (BSA and IgM). These
results indicate
that the antibody and lectin binding partners can immobilize hematopoietic
stem cells.
25
Example 3
IDENTIFICATION OF HEMATOPOIETIC STEM CELLS USING AN
ANTI-CARBOHYDRATE ANTIBODY
This Example illustrates the use of a representative antibody binding
30 partner to identify hematopoietic stem cells by fluorescence activated cell
sorting
(FACS).
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
18
The expression of markers on the surface of hematopoietic stem cells
was analyzed by a fluorescence activated cell sorter using red and green
fluorescence.
Human cord blood was washed twice with Dulbecco's PBS containing 0.1% sodium
azide, pH 7.4. The cells were then divided into separate tubes. Monoclonal
antibody
NUH2 and a control IgM were each added to a different tube at l0ug/ml. After
incubation at 4°C for 1 hour, the cells were washed and incubated with
FTTC-labeled
goat anti-mouse IgM and FITC-labeled streptavidin (lp,g/ml) respectively (KPL
Labs,
Gaithersburg, MD). After incubation at 4°C for 1 hour, the cells were
washed twice
with DPBS + azide and the commercial PE-labeled (red fluorescence) anti-CD34
10 antibody, HPCA2-PE (Becton Dickinson, San Jose, CA) was added to each tube.
After
incubation for 30 minutes at 4°C, the cells were washed and the red
blood cells were
lysed by the reagent ACK Lysing Buffer (Biofluids, Rockville, MD). Cells in
each tube
were then fixed by the addition of Formalin.
The results are presented in Figures 4 and 5, which display data obtained
15 from light scattered by lymphocytes in human blood from umbilical cords.
The data are
presented as contour plots which represent the bivariate distribution of green
and red
fluorescence. Contour lines known as isopleths are drawn to express a cell
count at a
particular set number. The X-axis is a logarithmic scale of green fluorescence
labeled
on cell surfaces, whereas the Y-axis is a logarithmic scale of red
fluorescence labeled on
20 cell surfaces. Quadrant 2 contains data that statistically demonstrate the
presence of
cells that express both surface markers, each detected by either the red or
green
fluorescent labeled antibody.
As shown in Figure 4, green fluorescent antibody NUH2 binds to almost
all CD34+ cells that are identified by the red fluorescent antibody HPCA2-PE.
Control
25 IgM antibodies did not label these cells (Figure S).
Example 4
IDENTIFICATION OF PLANT LECTIN BINDING PARTNERS
This Example illustrates the evaluation of representative lectin binding
30 partners.
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
19
Plant Iectins that bind CD34+ cells were also identified by two color
FACS analysis. Using the method described above the cells in human cord blood
were
labeled with green fluorescence-labeled plant lectins followed by the red
fluorescence-
labeled anti-CD34 antibody, HPCA2-PE. Results of the scatter diagrams are
summarized in Table 1 below. The binding of lectins to CD34+ cells is
consistent with
the expression of sialylated lactosamine structures on CD-34+ cells.
Table 1
Reactivity of Plant Lectins with Hematopoietic Stem Cells
Lectin Specificity Reactivity
Maackia amurensis NeuAcoc2-3Ga1 +
Sambucus nigra NeuAcoc2-6Ga1 +
Triticum vulgaris (WGA) NeuAc, GIcNAc(31-4GlcNAc +
Lycopersicon esculentum (Gal(31-4GlcNAc)", polylactosamine+
(tomato)
Erytheina cristagalli Gal(31-4GlcNAc +
(ECA)
Ulex europaeus (UEA) a-Fucose -
Lotus tetragonolobus a-Fucose -
Arachis hypogea (PNA) Ga1~31-3GalNAc
The positive results indicate that these lectin binding partners bind
sialylated lactosamine structures.
Example 5
DETECTION OF CELL SURFACE ANTIGENS ON CULTURED CELLS BY
IMMUNOFLUORESCENCE STAINING AND FLUORESCENCE
ACTIVATED CELL SORTING (FACS)
This Example illustrates the use of antibodies to detect cell surface
structures on a variety of cultured cell lines by immunofluorescent staining
followed by
fluorescence activated cell sortings (FACS).
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
20
The expression of markers on the surface of cultured cell lines was
analyzed by indirect immunofluorescent staining of the cell lines by specific
monoclonal antibodies followed by fluorescence activated cell sorting. The
following
cell lines were obtained from the ATCC (American Type Culture Collection,
Manassas,
5 VA). The ATCC designation follows the cell type abbreviation: KGIa,
CCL246.1;
HL60, CCL240; NCI-H446, HTB171; NCI-H82, HTB175; MCF7, HTB22 and Molt4,
CRL1582. Adherent cell lines were removed from tissue culture surfaces and
disaggregated by pipetting in 1X Versene (PBS-EDTA solution, Gibco, Life
Technologies, Grand Island, NY). Cells were washed in phosphate-buffered
saline
10 (PBS) and resuspended in Dulbecco's PBS (DPBS) containing 0.1% sodium azide
and
1% bovine serum albumin (BSA). The cells were then divided into 3 separate
tubes.
Monoclonal antibodies NUH2, anti-Lex, and a control mouse IgM were each added
to
separate tubes at final concentration of lOp,g/ml. After incubation at
4°C for 1 hour, the
cells in each tube were washed with DPBS + azide and incubated with FITC-
labeled
15 goat anti-mouse IgM (KPL Labs, Gaithersburg, MD) at a final concentration
of 1 ~tg/ml.
After incubation for 45 minutes at 4°C, the cells were washed twice
with DPBS + azide ,
and resuspended in DPBS + azide and 1% BSA. Labeled cells were maintained at
4°C
and analyzed immediately by fluorescence activated cell sorting gated for
fluorescein.
Cells were directly immunostained for antigens, CD34 and Thy-1 using
20 fluorescence-labeled antibodies anti-HPCA2-FITC and anti-Thy-1-PF,
respectively.
For these two antibodies, cells prepared as described above were incubated at
4°C for 1
hour in DPBS containing O.I% sodium azide, 1% BSA and 10 pg/mi of staining
antibody for either CD34 (anti-HPCA2-FTTC, Becton-Dickenson, San Jose, CA) or
Thy-1 (anti-CDw90-PE, Serotec, U.K.). After incubation, the cells were washed
twice
25 with DPBS + azide and resuspended in DPBS + azide and 1% BSA. Labeled cells
were
maintained at 4°C and analyzed immediately by fluorescence activated
cell sorting
gated for either fluorescein or phycoerythrin, respectively.
The results are presented in Table 2 below. Each of the antibodies
detects large differences in antigen presentation among the different cell
lines. Both
30 Thyl and the anti-carbohydrate antibody, anti-Lex [Gal(31-4(Fucocl-
2)GIcNAc~1-
3Gal~i1-R] bind to cell lines differently than the anti-carbohydrate antibody
NUH2
CA 02304961 2000-03-27
WO 99/15628 PCT/US98/20063
21
(NeuAca2-3Ga1(31-R]. In particular, KGIa cells, which express high levels of
CD34 on
their surfaces, are clearly devoid of the carbohydrate marker for
hematopoietic stem
cells, NeuAca2-3Ga1(31-4-R, detected by antibody NUH2. Thus, the hematopoietic
stem cell carbohydrate marker, NeuAca2-3Gal~i1-4-R is clearly different than
the
5 classic antigen for hematopoietic stem cells, CD34.
Table 2
FACS Analysis of Cell Lines with Various Antibodies (% positive)
Antibody KGIa HL-60 NCI-H446NCI-H82MCF7 Molt4
CD34 99 <5 <5 <5 <5 23
anti-Lex 17 65 75 <5 60 31
NUH2 <5 <5 25 <5 <5 <5
Thy-1 <5 <5 39 35 <5 <5
10 From the foregoing it will be appreciated that, although specific
embodiments of the invention have been described herein for purposes of
illustration,
various modifications may be made without deviating from the spirit and scope
of the
invention. Accordingly, the invention is not limited except as by the appended
claims.
