Note: Descriptions are shown in the official language in which they were submitted.
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Title: Method for Separating Cells Using Immunorosettes
FIELD OF THE INVENTION
The present invention relates to methods for separating cells using
imrnunorosettes. The invention includes novel antibody compositions for use in
the method
of the invention.
BACKGROUND OF THE INVENTION
In many applications it is desirable to enrich or, alternatively, deplete
certain cell populations in a biological sample. The fields of hematology,
immunology and
oncology rely on samples of peripheral blood and cell suspensions from related
tissues such
as bone marrow, spleen, thymus and fetal liver. The separation of specific
cell types from
these heterogeneous samples is key to research in these fields, diagnostics
and therapy for
certain malignancies and immune /hematopoietic disorders.
Purified populations of immune cells such as T cells and antigen presenting
cells are necessary for the study of immune function and are used in
immunotherapy.
Investigation of the cellular, molecular and biochemical processes require
analysis of
certain cell types in isolation. Numerous techniques have been used to isolate
T cell subsets,
B cells, basophils, NK cells and dendritic cells.
The isolation of hematopoietic stem cells has also been an area of great
interest. Pure populations of stem cells will facilitate studies of
hematopoiesis and
transplantation of hematopoietic cells from peripheral blood and/or bone
marrow is
increasingly used in combination with high-dose chemo- and/or radiotherapy for
the
treatment of a variety of disorders including malignant, nonmalignant and
genetic
disorders. Very few cells in such transplants are capable of long-term
hematopoietic
reconstitution, and thus there is a strong stimulus to develop techniques for
purification of
hematopoietic stem cells. Furthermore, serious complications and indeed the
success of a
transplant procedure is to a large degree dependent on the effectiveness of
the procedures
that are used for the removal of cells in the transplant that pose a risk to
the transplant
recipient. Such cells include T lymphocytes that are responsible for graft
versus host
disease (GVHD) in allogenic grafts, and tumor cells in autologous transplants
that may
cause recurrence of the malignant growth. It is also important to debulk the
graft by
removing unnecessary cells and thus reducing the volume of cyropreservant to
be infused.
In certain instances it is desirable to remove or deplete tumor cells from a
biological sample, for example in bone marrow transplants. Epithelial cancers
of the
bronchi, mammary ducts and the gastrointestinal and urogenital tracts
represent a major
type of solid tumors seen today. Micrometastatic tumor cell migration is
thought to be an
important prognostic factor for patients with epithelial cancer (Braun et al.,
2000;
Vaughan et al., 1990). The ability to detect such metastatic cells is limited
by the
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effectiveness of tissue or fluid sampling and the sensitivity of tumor
detection methods. A
technique to enrich circulating epithelial tumor cells in blood samples would
increase the
ability to detect metastatic disease and facilitate the study of such rare
cells and the
determination of the biological changes which enable spread of disease.
Hematopoietic cells and immune cells have been separated on the basis of
physical characteristics such as density and on the basis of susceptibility to
certain
pharmacological agents which kill cycling cells. The advent of monoclonal
antibodies
against cell surface antigens has greatly expanded the potential to
distinguish and
separate distinct cell types. There are two basic approaches to separating
cell populations
from blood and related cell suspensions using monoclonal antibodies. They
differ in
whether it is the desired or undesired cells which are distinguished/labelled
with the
antibody(s).
In positive selection techniques the desired cells are labelled with
antibodies and removed from the remaining unlabelled/unwanted cells. In
negative
selection, the unwanted cells are labelled and removed. Antibody/complement
treatment
and the use of immunotoxins are negative selection techniques, but FACS
sorting and most
batch wise irnmunoadsorption techniques can be adapted to both positive and
negative
selection. In immunoadsorption techniques cells are selected with monoclonal
antibodies
and preferentially bound to a surface which can be removed from the remainder
of the cells
e,g. column of beads, flasks, magnetic particles. Immunoadsorption techniques
have won
favour clinically and in research because they maintain the high specificity
of targeting
cells with monoclonal antibodies, but unlike FACSorting, they can be scaled up
to deal
directly with the large numbers of cells in a clinical harvest and they avoid
the dangers of
using cytotoxic reagents such as immunotoxins, and complement. They do
however, require
the use of a "device" or cell separation surface such as a column of beads,
panning flask or
magnet.
Current techniques for the isolation of hematopoietic stem cells, immune
cells and circulating epithelial tumor cells all involve an initial step to
remove red cells
then antibody mediated adherence to a device or artificial particle. (Firat et
al., 1988; de
Wynter et al., 1975; Shpall et al., 1994; Thomas et al., 1994; Miltenyi Biotec
Inc.,
Gladbach, Germany) In the case of positive selection there is yet another
step; removal of
the cells from the device or particle. All these multiple steps require time
and incur cell
loss. Slaper-Cortenbach et al. (1990) describes a method for purging bone
marrow of common
acute leukemic (cALL) cells using immunorosetting. The method requires that
the
erythrocytes are first removed from the bone marrow sample and are labelled
with
antibodies that bind to the cALL cells. The labelled erythrocytes are then
added back to
the sample where the cALL cells are immunorosetted. The depletion method works
best
when followed by an additional step of complement mediated lvsis of the cALL
cells.
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Density Separations are commonly used to isolate peripheral blood
mononuclear cells from granulocytes and erythrocytes. Ficoll (Amersham
Pharmacia
Biotech AB, Uppsala Sweden) is the most popular density solution used for this
application. In a Ficoll density separation whole blood is layered over
Ficoll, and then
centrifuged. The erythrocytes and granulocytes settle to the cell pellet and
the
mononuclear cells remain at the Ficoll plasma interface. The success of this
technique relies
on the difference in density between mononuclear cells and granulocytes. If
whole blood is
stored for more than 24 hours the granulocytes change density and will not
pellet with the
red cells. Suspensions of pure mononuclear cells can not be obtained from
stored blood or
samples with altered cell density in a single density separation.
In view of the foregoing, there is a need in the art to provide novel methods
for separating desired cells or removing unwanted cells from biological
samples.
SUMMARY OF THE INVENTION
The present inventors have developed a method for separating cells by
inununorosetting the cells with red blood cells or erythrocytes already
existing in the
sample. The method of the invention is a much simpler yet equally efficient
inununoaffinity technique as compared to the prior art methods. There is no
"device" or
_ need for an artificial separation surface (e.g., magnetic particles,
affinity column) not
normally present in the cell suspension. There is no need to first remove the
erythrocytes
from the sample and to then re-introduce them once they have been labelled
with
antibodies. Specific cell types are cross-linked to autologous erythrocytes
found within the
sample and subsequent rosettes are then removed by sedimentation or
centrifugation.
Accordingly, in one embodiment, the present invention provides a method of
separating nucleated cells from a sample comprising the nucleated cells and
erythrocytes
comprising:
(1) contacting the sample with an antibody composition comprising (a) at
least one antibody that binds to an antigen on the nucleated cells to be
separated linked, either directly or indirectly, to (b) at least one antibody
that binds to the erythrocytes, under conditions to allow immunorosettes of
the nucleated cells and the erythrocytes to form; and
= (2) removing the immunorosettes from the sample.
The method can be used in both positive and negative selection protocols.
The method can be used on any sample that contains red blood cells including
whole blood,
bone marrow, fetal liver, cord blood, buffy coat suspensions, pleural and
peritoneal effusion
and samples of thymocytes and splenocytes.
=
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BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
Figure 1 is a schematic diagram of a rosette of erythrocytes formed around
an unwanted nucleated cell using tetrameric antibody complexes.
DETAILED DESCRIPTION OF THE INVENTION
I. METHOD OF THE INVENTION
As hereinbefore mentioned, the present invention relates to a method for
separating cells by immunorosetting the cells with red blood cells.
In its broadest aspect, the present invention provides a method of
separating nucleated cells from a sample comprising the nucleated cells and
erythrocytes
comprising:
(1) contacting the sample with an antibody composition comprising (a) at
least one antibody that binds to an antigen on the nucleated cells to be
separated linked, either directly or indirectly, to (b) at least one antibody
that binds to the erythrocytes, under conditions to allow immunorosettes of
the nucleated cells and the erythrocytes to form; and
=
(2) removing the immunorosettes from the sample.
The method can be used in both positive and negative selection protocols. In
.
positive selection, the desired cells are rosetted. In such an embodiment, the
method would
further include the step of lysing the red blood cells in the immunorosettes
and separating
the desired cells. Accordingly, in a positive selection method the antibody
composition
will contain (a) at least one antibody specific for the nucleated cells that
one wishes to
obtain or separate from the sample.
Preferably, the method of the invention is used in a negative selection
protocol. In negative selection, the desired cells are not immunorosetted and
would be
remaining in the sample once the immunorosettes have been removed. In a
negative
selection method, the antibody composition will contain (a) at least one
antibody specific
for the cells that one wishes to remove from the sample. Accordingly, the
present invention
provides a negative selection method for enriching and recovering desired
cells in a sample
containing the desired cells, erythrocytes and undesired cells comprising:
" (1) contacting the sample with an antibody composition comprising (a) at
least one antibody that binds to an antigen on the undesired cells
linked, either directly or indirectly, to (b) at least one antibody that .
binds to the erythrocytes, under conditions to allow immunorosettes of
the undesired cells and the erythrocytes to form; and
(2) separating the immunorosettes from the remainder of the sample to
obtain a sample enriched in the desired cells.
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The immunorosettes between the erythrocytes and the unwanted cells
formed in step (1) can be separated from the desired cells using a variety of
techniques. In
one embodiment, the sample, containing the immunorosettes, is layered over a
buoyant
density solution (such as Ficoll-Hypaque) and centrifuged. The immunorosettes
pellet and
the desired cells remain at the interface between the buoyant density solution
and the
sample. The desired cells are then removed from the interface for further use.
In another
embodiment, the sample containing the immunorosettes obtained in step (1) is
mixed with a
sedimentation reagent (such as hydroxyethyl starch, gelatin or methyl
cellulose) and the
rosettes are permitted to sediment. The desired cells remain in suspension and
are removed
for further use. In a further embodiment, the sample containing the
immunorosettes
obtained in step (1) is allowed to sediment with or without the aid of
centrifugation or
Counter Flow Elutriation. The desired cells remain in suspension and are
removed for
further use.
The antibody compositions for use in the present invention are described in
greater detail below.
The method of the invention may be used in the processing of biological
samples that contain erythrocytes including blood (in particular, cord blood
and whole
blood) bone marrow, fetal liver, buffy coat suspensions, pleural and
peritoneal effusions and
suspensions of thymocytes and splenocytes. Surprisingly, the inventors have
found that the
method can be used to remove cells directly from whole blood or whole bone
marrow
without prior processing. This offers a significant advantage of the method of
the
invention over the prior art methods. In particular, the erythrocytes do not
have to be
removed, labelled and added back to the sample.
The method of the invention can be used to prepare enriched samples of any
cell type including, but not limited to, T cells, B cells, NK cells, dendritic
cells, monocytes,
basophils, mast cells, progenitor cells, stem cells and tumor cells.
In one embodiment, the method of the invention can be used to prepare
hematopoietic progenitor and stem cell preparations from bone marrow samples.
For
example, the method of the invention may be used in a negative selection
protocol to
deplete or purge B and T lymphocytes, monocytes, NK cells, granulocytes,
and/or tumor
cells from samples to prepare hematopoietic progenitor and stem cell
preparations for use in
transplantation as well as other therapeutic methods that are readily apparent
to those of
skill in the art. For example, bone marrow or blood can be harvested from a
donor in the
case of an allogenic transplant and enriched for progenitor and stem cells by
the method
described herein. Using negative selection the human hematopoietic progenitor
and stem
cells in the preparation are not coated with antibodies, or modified making
them highly
suitable for transplantation and other therapeutic uses that are readily
apparent to those
skilled in the art.
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In another embodiment, the method of the invention can be used to isolate
and recover mature dendritic cells and their precursors from blood. Dendritic
cells have
many useful applications including as antigen presenting cells capable of
activating T cells
both in vitro and in vivo. As an example, dendritic cells can be loaded
(pulsed) in vitro
with a tumor antigen and injected in vivo to induce an anti-tumor T cell
response.
In a further embodiment, the method of the invention may also be used to
prepare a cell preparation from samples such as blood and bone marrow, which
is enriched
in a selected differentiated cell type such as T-cells, B-cells, NK cells,
monocytes, dendritic
cells, basophils and plasma cells. This will enable studies of specific cell
to cell
interactions including growth factor production and responses to growth
factors. It will also
allow molecular and biochemical analysis of specific cells types. Cell
preparations
enriched in NK cells, dendritic cells and T-cells may also be used in irrunune
therapy
against certain malignancies.
In yet another embodiment, the method of the invention can be used to
separate tumor cells, such as non-hematopoietic metastatic tumor cells from a
sample. The
method is useful in the detection of non-hematopoietic tumor cells from blood,
bone marrow,
and peritoneal and pleural effusions of patients to aid in the diagnosis and
detection of
metastatic disease, monitoring the progression of metastatic disease, or
monitoring the
efficacy of a treatment.
II. ANTIBODY COMPOSITIONS
The invention includes the antibody compositions for use in the method of
the present invention. The antibody composition will contain (a) at least one
antibody that
binds to an antigen on nucleated cells linked, either directly or indirectly,
to (b) at least one
antibody that binds to an antigen on erythrocytes.
The term "at least one antibody" means that the antibody composition
includes at least one type of antibody (as opposed to at least one antibody
molecule). One
type of antibody means an antibody that binds to a particular antigen. For
example,
antibodies that bind to the antigen CD2 are considered one type of antibody.
Preferably,
the antibody compositions of the invention contain (a) more than one antibody
type that
binds to nucleated cells.
The two antibodies (a) and (b) may be = directly linked by preparing
bifunctional or bispecific antibodies. The two antibodies (a) and (b) may be
indirectly
linked for example, by preparing tetrameric antibody complexes. All of these
are described
hereinafter.
In one aspect, the antibody specific for the nucleated cells is linked
directly
to the antibody specific for the erythrocytes. In one embodiment, the antibody
composition
of the present invention contains bifunctional antibodies comprising at least
one antibody
specific for the nucleated cells linked directly to (b) at least one antibody
specific for the
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erythrocytes. Bifunctional antibodies may be prepared by chemically coupling
one
antibody to the other, for example by using N-succinimidy1-3-(2-pyridyldithio)
propionate (SPDP).
In another embodiment, the antibody composition contains bispecific
antibodies. Bispecific antibodies contain a variable region of an antibody
specific for
erythrocytes and a variable region specific for at least one antigen on the
surface of the
nucleated cells to be separated. The bispecific antibodies may be prepared by
forming
hybrid hybridomas. The hybrid hybridomas may be prepared using the procedures
known
in the art such as those disclosed in Staerz & Bevan, (1986, PNAS (USA) 83:
1453) and
Staerz & Bevan, (1986, Immunology Today, 7:241). Bispecific antibodies may
also be
constructed by chemical means using procedures such as those described by
Staerz et al.,
(1985, Nature, 314:628) and Perez et al., (1985 Nature 316:354), or by
expression of
recombinant immunoglobulin gene constructs.
In another aspect, the antibody composition of the present invention
comprises (a) at least one antibody specific for a nucleated cell type
indirectly linked to (b)
at least one antibody specific for the erythrocyte. By "indirectly linked" it
is meant that
antibody (a) and antibody (b) are not directly covalently linked to each other
but are
attached through a linking moiety such as an immunological complex. In a
preferred
embodiment, the antibody to the nucleated cell type is indirectly linked to
the antibody
specific for the erythrocytes by preparing a tetrameric antibody complex. A
tetrameric
antibody complex may be prepared by mixing a first monoclonal antibody which
is capable
of binding to the erythrocytes, and a second monoclonal antibody capable of
binding the
nucleated cells to be separated. The first and second monoclonal antibody are
from a first
animal species. The first and second antibody are reacted with approximately
an
equimolar amount of monoclonal antibodies. of a second animal species which
are directed
against the Fc-fragments of the antibodies of the first animal species. The
first and second
antibody may also be reacted with an about equirnolar amount of the F(a1:)2
fragments of
monoclonal antibodies of a second animal species which are directed against
the
Fc-fragments of the antibodies of the first animal species. (See U.S. Patent
No. 4,868,109 to
Lansdorp, for a description of tetrameric antibody complexes and methods for
preparing
same).
Preferably, the antibody specific for the erythrocytes is anti-glycophorin
A. The anti-glycophorin A antibodies contained in the antibody composition of
the
invention are used to bind the erythrocytes. Examples of monoclonal antibodies
specific for
glycophorin A are 10F7MN (U.S. Patent No. 4,752,582, Cell lines: ATCC
accession numbers
HB-8162), and D2.10 (Immunotech, Marseille, France).
Preferably, the antibody specific for the nucleated cells is a combination of
antibodies. The combination of antibodies may be specific for a number of cell
types so that
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many cell types may be removed from the sample. When using a combination of
antibodies,
each antibody will be linked (either directly or indirectly) to an antibody
specific for
erythrocytes.
In a preferred embodiment, the antibody composition is a tetrameric
complex comprising (a) anti-glycophorin A antibodies to bind the erythrocytes,
(b) an
antibody that binds to a nucleated cell type that one wishes to immunorosette
and (c)
antibodies that bind the Fc portion of both (a) and (b), optionally F(ab')2
antibody
fragments. The molar ratio of (a):(b):(c) may be approximately 1:3:4. When
several types
of cells are to be separated, complexes are made with several anti-nucleated
cell antibodies
(b). The complexes may then be mixed together to form an antibody composition
for use in
the method of the invention. Figure 1 is a schematic diagram of a rosette
formed by
tetrameric antibody complexes.
Within the context of the present invention, antibodies are understood to
include monoclonal antibodies and polyclonal antibodies, antibody fragments
(e.g., Fab,
and F(ab')2), chimeric antibodies, bifunctional or bispecific antibodies and
tetrameric
antibody complexes. Antibodies are understood to be reactive against a
selected antigen on
the surface of a nucleated cell or erythrocyte if they bind with an
appropriate affinity
(association constant), e.g. greater than or equal to 107 M-1.
Monoclonal antibodies are preferably used in the antibody compositions of
the invention. Monoclonal antibodies specific for selected antigens on the
surface of
nucleated cells may be readily generated using conventional techniques. For
example,
monoclonal antibodies may be produced by the hybridoma technique originally
developed
by Kohler and Milstein 1975 (Nature 256, 495-497; see also U.S. Patent Nos. RE
32,011,
4,902,614, 4,543,439, and 4,411,993; see also Monoclonal Antibodies,
Hybridomas: A
New Dimension in Biological Analyses, Plenum Press, Kennett, McKeam, and
Bechtol
(eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.),
Cold Spring
Harbor Laboratory Press, 1988). Other techniques may also be utilized to
construct
monoclonal antibodies (for example, see William D. Huse et al.,
1989,"Generation of a
Large Combinational Library of the Immunoglobulin Repertoire in Phage Lambda,"
Science 246: 1275-1281, L. Sastry et al., 1989 "Cloning of the Immunological
Repertoire
in Escherichia coli for Generation of Monoclonal Catalytic Antibodies:
Construction of a
Heavy Chain Variable Region-Specific cDNA Library," Proc Natl. Acad. Sci USA
86:
5728-5732; Kozbor et al., 1983 Immunol. Today 4,72 re the human B-cell
hybridoma
technique; Cole et al. 1985 Monoclonal Antibodies in Cancer Therapy, Allen R.
Bliss, Inc.,
pages 77-96 re the EBV-hybridoma technique to produce human monoclonal
antibodies;
and see also Michelle Alting-Mees et al., 1990 "Monoclonal Antibody Expression
Libraries: A Rapid Alternative to Hybridomas," Strategies in
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Molecular Biology 3:1-9). Hybridoma cells can be screened immunochemically for
production of antibodies specifically reactive with an antigen, and monoclonal
antibodies
can be isolated.
Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described above for whole
antibodies.
For example, F(ab')2 fragments can be generated by treating antibody with
pepsin. The
resulting F(ab')2 fragment can be treated to reduce disulfide bridges to
produce Fab'
fragments.
The invention also contemplates chimeric antibody derivatives, i.e.,
antibody molecules that combine a non-human animal variable region and a human
constant
region. Chimeric antibody molecules can include, for example, the antigen
binding domain
from an antibody of a mouse, rat, or other species, with human constant
regions. A variety
of approaches for making chimeric antibodies have been described and can be
used to make
chimeric antibodies containing the irrununoglobulin variable region which
recognizes
selected antigens on the surface of differentiated cells or tumor cells. See,
for example,
Morrison et al., 1985; Proc. Natl. Acad. Sci. U.S.A. 81,6851; Takeda et al.,
1985, Nature
314:452; Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent
No. 4,816,397;
Tanaguchi et al., European Patent Publication EP171496; European Patent
Publication
0173494, United Kingdom patent GB 2177096B.
Bifunctional antibodies may be prepared by chemical conjugation, somatic
hybridization or genetic engineering techniques.
Chemical conjugation is based on the use of homo- and heterobifunctional
reagents with e-amino groups or hinge region thiol groups. Homobffunctional
reagents such
as 5,5'-Dithiobis(2-nitrobenzoic acid)(DNTB) generate disulfide bonds between
the two
Fabs, and 0-phenylenedimaleimide (0-PDM) generate thioether bonds between the
two
Fabs (Brenner et al., 1985, Glennie et al., 1987). Heterobifunctional reagents
such as N-
succinimidy1-3-(2-pyridylditio) propionate (SPDP) combine exposed amino groups
of
antibodies and Fab fragments, regardless of class or isotype (Van Dijk et al.,
1989).
Somatic hybridization includes fusion of two established hybridomas
generating a quadroma (Milstein and Cuello, 1983) or fusion of one established
hybridoma
with lymphocytes derived from a mouse immunized with a second antigen
generating a
trioma (Nolan and Kennedy, 1990). Hybrid hybridomas are selected by making
each
hybridoma cell line resistant to a specific drug-resistant marker (De Lau et
al., 1989), or by
labeling each hybridoma with a different flucirochrome and sorting out the
heterofluorescent cells (Karawajew et al., 1987).
Genetic engineering involves the use of recombinant DNA based technology
to ligate sequences of DNA encoding specific fragments of antibodies into
plasmids, and
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expressing the recombinant protein. Bispecffic antibodies can also be made as
a single
covalent structure by combining two single chains Fv (scFv) fragments using
linkers (Winter
and Milstein, 1991); as leucine zippers coexpressing sequences derived from
the transcription
factors fos and jun (Kostelny et al., 1992); as helix-turn-helix coexpressing
an interaction
domain of p53 (Rheinnecker et al., 1996), or as diabodies (Holliger et al.,
1993).
Table 1 provides examples of antibodies to particular human antigens on
nucleated cells that may be used in the method of the present invention. The
method of the
invention may also be used for other species. The choice of the antibody or
antibodies to the
nucleated cells will depend on the nature of the sample, the choice of the
cells to be
enriched or depleted and whether the method is a positive or negative
selection protocol.
In all cases, the antibody (or antibodies) to the nucleated cells to be
immunorosetted will be
linked, either directly or indirectly, to the antibody specific for the
erythrocytes when
used in the method of the invention.
The methods and antibody compositions of the invention are preferably
used in negative selection protocols to prepare a cell preparation which is
enriched for a
specific cell type. This is achieved by using antibody compositions which lack
antibodies
to the specific cell type that you wish to isolate. Accordingly, the present
invention
provides an antibody composition for enriching and recovering desired cells in
a sample
containing desired cells, erythrocytes and undesired cells comprising (a) at
least one
antibody that binds to an antigen on the undesired cells linked to (b) at
least one antibody
that binds to the erythrocytes. Particular embodiments of the antibody
compositions that
may be used in negative selection protocols of the invention for human cells
are set out in
Table 2. This Table provides a list or cocktail of antibodies to particular
antigens that can
be used as antibody (a) in the above method to enrich for a particular cell
type. In most
cases, several choices for the essential antibodies are provided as well as
several optional
antibodies. For example, for enriching for T cells antibody (a) may be a
cocktail of
antibodies to (1) CD16 and/or CD66b and/or CD11b and/or CD15; (2) CD19 and/or
CD20
and/or CD21 and/or CD22 and/or CD24 and/or Ig; and (3) CD36 and/or CD14. The
cocktail
may optionally include antibodies to CD33 and/or CD56 and/or IgE and/or CD41.
In
addition to the antibody combinations listed in Table 2, one skilled in the
art will
appreciate that other antibody combinations may be used to enrich for specific
cell types
such as those described in U.S. Patent No. 5,877,299,
As the invention relates to the preparation of immunorosettes to prepare
enriched cell preparations, one skilled in the art will appreciate that other
antibodies and
=
antibody combinations may be used. =
The methods and antibody compositions of the invention may be used in
= positive selection protocols to prepare a cell preparation in which the
desired cells are
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immunorosetted. Some examples of antibody combinations useful in positive
selection
protocols are set out below.
To separate non-hematopoietic tumor cells in a positive selection protocol,
the antibody composition includes antibodies specific for non-hematopoietic
antigens
expressed on tumor cells, such as antibodies against antigens expressed on the
surface of
breast and lung carcinoma and neuroblastoma cells. The antibodies to the non-
hematopoietic antigens expressed on epithelial tumor cells may be obtained
from
commercial sources (for example as shown in Table 3) or prepared using
techniques known in
the art.
To separate B cells in a positive selection protocol, the antibody
composition contains antibodies against CD24 and/or CD19 and/or CD20 and/or
CD22.
To separate T cells in a positive selection protocol, the antibody
composition contains antibodies against CD3 and/or CD2 and/or CD5 and/or both
CD4 and
CD8.
= To separate NK cells in a positive selection protocol, the antibody
composition contains antibodies against CD56.
To separate granulocytes in a positive selection protocol, the antibody
composition contains antibodies against CD16 and/or CD66e and/or CD66b.
= To separate monocytes in a positive selection protocol, the antibody
composition contains antibodies against CD14.
The following non-limiting examples are illustrative of the present
invention:
EXAMPLES
Example' =
Preparation of Tetramers
In order to prepare a tetrameric antibody complex for use in the method of
the present invention, the following protocol may be used: (a) take 1 mg of
antibody
specific for cells to be rosetted (e.g. anti-CD2, CD3, CD4, CD8, CD14, CD16,
CD19 etc.); (b)
add 3 mg anti-Glycophorin A antibody (against red blood cells); mix well (c)
then add 4.0
mg of P9 antibody or 2.72 mg of the P9 F(abs)2 antibody, fragment. Incubate
overnight at
37 C. The P9 antibody binds the Fc portion of the antibodies added in steps
(a) and (b)
resulting in a tetraMeric antibody complex. For more information on the
preparation of
tetramers see 11.S. Patent No. 4,868,109 to Lansdorp.
Tetrameric antibody complexes incorporating different antibodies to antigens
expressed or nudeated cells are prepared separately and then mixed.
The antibody compositions are made by combining various tetrameric
antibody complexes depending on which cells one wishes to deplete. The
concentration of
the various tetrameric antibody complexes varies: typically antibodies to
antigens
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expressed on nucleated cells are at 10-301.1g/mL in tetrameric complexes. The
composition is
then diluted 1/10 into the cells so the final concentrations of each anti
nucleated cell
antibody in the cell suspensions is 1.0-3.0 p.g/mL.
Example 2
Method of Inununorosetting Using Ficoll
A negative selection protocol for immunorosetting cells from whole
peripheral blood using Ficoll Hypaque is set out below.
1. Add 1004 antibody composition per mL of whole peripheral
blood.
2. Incubate 20 minutes at room temperature.
3. Dilute sample with an equal volume of phosphate buffered saline
(PBS) + 2% fetal calf serum (FCS) and mix gently.
4. Layer the diluted sample on top of Ficoll Hypaque or layer the Ficoll
underneath the diluted sample.
5. Centrifuge for 20 minutes at 1200 x g, room temperature, with the brake
= off.
6. Remove the enriched cells from the Ficoll:plasma interface.
7. Wash enriched cells with 5-10x volume of PBS + 2% FBS.
Note: For enrichment of monocytes and other adherent cells, add 1 mM EDTA to
the sample
of whole blood and to all wash/dilution solutions.
Example 3
Method of Immunorosetting Using Hetastarch Sedimentation
A negative selection protocol for immunorosetting cells from whole
peripheral blood using hetastarch is set out below. Hetastarch is one of a
number of
compounds that increases red blood cell sedimentation rates through
agglutination.
1. Add 1 mL of 6% hetastarch in saline per 5 mL of blood and mix.
2. Add antibody composition described in Example 1 to whole
blood such
that each anti-nucleated cell antibody is at a final concentration of
1.0-2.0 tg/mL.
3. Incubate 10 minutes at room temperature.
4. Centrifuge for 5 minutes at 50 x g, room temperature.
5. Remove supernatant. This fraction contains the enriched cells.
6. Wash enriched cell fraction with 2-5 x volume of PBS +2% fetal bovine
=
serum (FBS).
Example 4
Method of Inununorosetting Using Hetastarch/lodixanol Mixture
A negative selection protocol for immunorosetting cells from whole
peripheral blood is set out below.
1. Add 1 mL of 6% hetastarch in saline per 5 mL of blood and
mix.
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2. Add 0.6 mL of 60% w/v iodixanol and mix. Iodixanol is one of a number
of compounds that increases the aqueous solution density appreciably.
3. Add antibody composition described in Example 1 to whole blood such
that each anti-nucleated cell antibody is at a final concentration of
1.0-2.0 tig/mL.
4. Incubate 10 minutes at room temperature.
5. Centrifuge for 5 minutes at 50 xg, room temperature.
6. Remove supernatant. This fraction contains the enriched cells.
7. Wash enriched cell fraction with 2-5 x volume of PBS +2% FBS.
Example 5
Method of Immunorosetting - Positive Selection
A positive selection protocol for immunorosetting cells from whole
peripheral blood is set out below.
1. Set aside 1 mL of blood.
2. Layer 10 mL of blood over Ficoll-Paque and centrifuge for 20 minutes at
1200xg, room temperature, brake off.
3. Recover the MNC layer at the Ficoll:plasma interface, wash with
PBS+2`)/0 FBS.
4. Count cells and resuspend at 1x108/mL.
5. Measure sample volume, designated volume A.
6. Add 0.2 mL of reserved blood from Step 1.
7. Make up total volume to twice volume A with PBS+2 /0 FBS.
8. Add a tetrameric antibody complex specific to a given antigen at a
final concentration of 1.0 p.g/mL, the synthesis of which is described in
Example 1.
9. Incubate 20 minutes at room temperature.
10. Dilute by a factor of 2 with PBS+2`)/0 FBS and layer over Percoll
= prepared at a density of 1.085 g/mL and 'an osmolarity of 280 mOsm.
11. Centrifuge for 20 minutes at 1200xg as in Step 2.
12. Discard supernatant and resuspend pellet containing the enriched
cells.
13. Lyse red blood cells with ammonium chloride solution and wash with
PBS+2 /0 FBS.
Example 6
Enrichment of T cells - Immunorosetting Using Ficoll
This example demonstrates the enrichment of T cells from whole
peripheral blood using the method described in Example 2. A T cell enrichment
cocktail of
tetrameric antibody complexes containing antibodies against CD16, CD19, CD36
and CD56
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was prepared. The results, shown in Table 4, demonstrate that the method of
the invention
results in 95% purity of T cells with a recovery of close to 50%.
Example Z
Enrichment of CD8+ T cells - Immunorosetting Using Ficoll
This example demonstrates the enrichment of CD8 + T cells from whole
peripheral blood using the method described in Example 2. Two cocktails of
tetrameric
antibody complexes were tested. One cocktail contained antibodies against CD4,
CD16,
CD19, CD36 and CD56 the other contained antibodies to CD4, CD16, CD19, CD36,
CD56 and
IgE. The results, shown in Table 5, demonstrate that the addition of anti IgE
to the cocktail
improves the purity of CD8+ T cells with no effect on recovery.
Example 8
Enriclunent of CD4 + T cells - Immunorosetting Using Ficoll
This example demonstrates the enrichment of CD4+ T cells from whole
peripheral blood using the method described in Example 2. Two CD4 T cell
enrichment
cocktails of tetrameric antibody complexes were prepared. One cocktail
contained
antibodies to CD8, CD16, CD19, CD36 and CD56. The other cocktail contained
antibodies
to CD8, CD16, CD19, CD36, CD56 and IgE. The results, shown in Table 6,
demonstrate that
the method of the invention results in 93% purity of CD4+ T cells with a
recovery of 46%
and that addition of anti-IgE to the enrichment cocktail improves the purity
of CD4+ T
cells.
Example 9
Enrichment of 13 cells - Immunorosetting Using Ficoll
This example demonstrates the enrichment of B cells from whole
peripheral blood using the method described in Example 2. Two B cell
enrichment cocktails
of tetrameric antibody complexes were prepared. One cocktail contained
antibodies to CD2,
CD3, CD16, CD36 and CD56. The other cocktail contained antibodies to CD2, CD3,
CD16,
CD36, CD56. and IgE. The results, shown in Table 7, demonstrate that the
method of the
invention results in 88% purity of B cells with a recovery of 43% and that
addition of anti-
IgE to the cocktail improves the purity of 8 cells.
Example 10
Enrichment of NK cells - Inununorosetting Using Ficoll
= This example demonstrates the enrichment of NK cells from whole
peripheral blood using the method described in Example 2. Two NK cell
enrichment
cocktails of tetrameric antibody complexes were prepared. One cocktail
contained
antibodies to CD3, CD4, CD19, CD66b and CD36. The other cocktail contained
antibodies
to CD3, CD4, CD19, CD66b, CD36 and IgE. The results, shown in Table 8,
demonstrate that
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the method of the invention results in 74% purity of NK cells with a recovery
of 44% and
that the addition of anti-IgE to the cocktail improves purity but decreases
recovery.
Example 11
Enrichment of Progenitors
This example demonstrates the enrichment of progenitor cells from whole
umbilical cord blood using the method described in Example 2. Two different
cocktails of
tetrameric antibody complexes were used;
( a ) the progenitor enrichment cocktail containing tetrameric antibody
complexes to CD2, CD3, CD14, CD16, CD19, CD24, CD56 and CD66b;
(b) the de-bulking cocktail containing tetrameric antibody complexes to
CD2, CD14, CD19 and CD66b.
The results, shown in Table 9, demonstrate that the method of the invention
results in 29%
purity of CD34+ cells with a recovery of 53% for the extensive progenitor
enrichment
cocktail and only 5% purity and 45% recovery for the four antibody de-bulking
cocktail.
Example 12
Enrichment of Monocytes - Immunorosetting Using Ficoll
This example demonstrates the enrichment of monocytes from whole
peripheral blood using the method described in Example 2. Several monocyte
enrichment
cocktails of tetrameric antibody were prepared (see Table 10). The results
shown in Table
10 demonstrate that the method of the invention results in 76% purity of CD14
+ cells with
65% recovery of CD14 + cells and that the addition of anti CD8 or anti-IgE
improved the
purity of monocytes but adding both anti-CD8 and IgE did not have an additive
effect.
Example 13
Enrichment of Non-Hematopoietic Tumor Cells
This example demonstrates the enrichment of breast cancer cells from whole
peripheral blood using the method described in Example 2. Cells from the CAMA
breast
cancer cell line were seeded into samples of whole peripheral blood at a
frequency of 1/103,
1/104 and 1/105. Three tumor cell enrichment cocktails of tetrameric antibody
complexes
were prepared. The antibody composition of the cocktails is listed in Table
11. The results,
shown in Table 12, demonstrate that the method of the invention results in
greater than 2
log enrichment of tumor cells with 20-50% recovery of tumor cells. The more
extensive
cocktail offers a greater degree of tumor cell enrichment.
Example 14
T Cell Enrichment - Effect of Substituting Anti-CD14 with Anti-CD36
This example demonstrates the improved T cell enrichment from whole
peripheral blood using the method described in Example 2 when the enrichment
cocktail is
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modified by substituting anti-CD36 for anti-CD14. The results in Table 13 show
a 24%
increase in %purity of CD3 + cells with the antibody substitution.
Example 15
Enrichment of Specific Cell Populations Using Hetastarch Sedimentation
This example demonstrates the enrichment of various cell populations from
whole peripheral blood using the method described in Example 3.
A T cell enrichment cocktail of tetrameric antibody complexes containing
antibodies against CD16, CD19, CD36 and CD56 was prepared. The method of the
invention results in greater than 95% purity of T cells, with a recovery of
60%.
A B cell enrichment cocktail of tetrameric antibody complexes containing
antibodies against CD2, CD3, CD16, CD36 and CD56 was prepared. The method of
the
invention results in 75% purity of B cells, with a recovery of 39%.
A NK cell enrichment cocktail of tetrameric antibody complexes containing
antibodies against CD3, CD4, CD19, CD36 and CD66b was prepared. The method of
the
invention results in 65% purity of NK cells, with a recovery of 27%.
Example 16
Enrichment of Specific Cell Populations Using Hetastarch/lodixanol Mixture
This example demonstrates the enrichment of various cell populations from
whole peripheral blood using the method described in Example 4. The results,
listed in
Table 14, are summarized as follows.
A T cell enrichment cocktail of tetrameric antibody complexes containing
antibodies against CD16, CD19, CD36 and CD56 was prepared. The method of the
invention results in 95% purity of T cells, with a recovery of 61%.
A CD4 + T cell enrichment cocktail of tetrameric antibody complexes
containing antibodies against CD8, CD16, CD 19, CD36 and CD56 was prepared.
The
method of the invention results in 89% purity of CD4+ T cells, with a recovery
of 64%.
A CD8+ T cell enrichment cocktail of tetrameric antibody complexes
containing antibodies against CD4, CD16, CD 19, CD36 and CD56 was prepared.
The
method of the invention results in 80% purity of CD8+ T cells, with a recovery
of 43%.
A B cell enrichment cocktail of tetrameric antibody complexes containing
antibodies against CD2, CD3, CD16, CD36 and CD56 was prepared. The method of
the
invention results in 84% purity of B cells, with a recovery of 58%.
A NK cell enrichment cocktail of tetrameric antibody complexes containing
antibodies against CD3, CD4, CD 19, CD36 and CD66b was prepared. The method of
the
invention results in 80% purity of NK cells, with a recovery of 50%.
=
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Example 17
Immunorosetting Using Different Layering Media
This example demonstrates that the method of Example 2 can be modified
by substituting different media for Ficoll-Hypaque in Step 4. The density of
Ficoll was
1.077 g/mL and the osmolarity was approximately 300 mOsm. Percoll and
Iodixanol
solutions were prepared with a density of 1.085 g/mL and an osmolarity of 280
mOsm. A B
cell enrichment cocktail containing antibody complexes against CD2, CD3, CD16,
CD36 and
CD56 was prepared.
The results of B cell enrichments for two separate samples, shown in Table
15, demonstrate that the use of different layering media at a higher density
can increase
the recovery of B cells without lowering the B cell purity.
Example 18
Purging of T Cells Using Immunorosettes
This example demonstrates the removal of T cells from whole peripheral
blood using the method described in Example 2. T cells purging cocktail of
tetrameric
antibody complexes to CD3 was prepared. The method of the invention resulted
in 2.3 log
depletion of CD3 + cells.
Example 19
Purging of B Cells Using Immunorosettes
This example demonstrates the removal of B cells from whole peripheral
blood using the method described in Example 2. B cells purging cocktail of
tetrameric
antibody complexes to CD19 was prepared. The method of the invention resulted
in 3.0 log
depletion of CD19+ cells.
Example 20
Purging of Breast Carcinoma Cells Using hrununorosetting
This example demonstrates the removal of breast carcinoma cells from
whole peripheral blood seeded with 1-5% CAMA breast carcinoma cells using the
method
described in Example 2. A purging cocktail of tetrameric antibody complexes
containing
anti-breast carcinoma antibodies 5E11 and BRST 1 was prepared. The results
shown in
Table 16 demonstrate the method of the invention results in 1.0-1.4 log
depletion of breast
carcinoma cells.
Example 21
Removal of Granulocytes from Previously Stored Whole Peripheral Blood
The density of granulocytes in samples of whole peripheral blood decreases
with >24 hours of storage. Density separation methods conunonlv used to remove
red cells
and granulocytes from fresh whole blood do not efficiently remove
granulocy.tes from stored
blood samples. The sedimentation rate of stored granulocytes can be increased
to allow
efficient removal in a standard Ficoll density separation (1.077 g/mL) by
immunorosetting.
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This example demonstrates the removal of granulocytes from stored (48 hour)
whole
peripheral blood using the method described in Example 2. A granulocyte
depletion
cocktail containing tetrameric antibody complexes against CD66b was prepared.
The
results, shown in Table 17, demonstrate that the method of the invention
results in 1.8-2.6
log depletion of granulocytes.
Example 22
Positive Selection of Specific Cell Populations Using Immunorosetting
This example demonstrates the enrichment of CD8+ cells from whole
peripheral blood using the positive selection method described in Example 5. A
tetrameric
antibody complex against CD8 was prepared. The method of the invention results
in the
enrichment of CD8+ cells as a percentage of the mononuclear cell fraction from
25% in the
start to 32% in the pellet.
The scope of the claims should not be limited by the preferred embodiments
and examples, but should be given the broadest interpretation consistent with
the
description as a whole.
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TABLE 1
Antibodies used in Cell Separation
Antigen Antibody l Source
CCR5 BLR-7 R&D, Minneapolis, MN
CD2 6F10.3 IMMUNOTECH, Marseille, France
MT910 Dako, Carpinteria, CA
CD3 UCHT1 IMMUNOTECH, Marseille, France
SK 7 Becton Dickinson Immunocytometry, Mountain View,
Calif.
CD4 13B8.2 Becton Dickinson lmmunocytometry, Mountain View,
Calif.
CD5 UCHT2 Serotec, Raleigh, NC
CD8 B911 Becton Dickinson laununocytometry, Mountain View,
Calif.
= OKT3 BioDesigns
CD10 ALB1 IMMUNOTECH, Marseille, France
CD1lb ICRF44 Pharmingen, San Diego, CA
CD14 MEM 15 Exbio, Praha, Czech Republic
MEM 18
CD15 DU-HL60-3 Sigma, St. Louis, MO
CD16 MEM 154 Exbio, Praha, Czech Republic
3G8 IMMUNOTECH, Marseille, France
NKP15 Becton Dickinson Imrnunocytometry, Mountain View,
Calif.
CD19 J4.119 IMMUNOTECH, Marseille, France
4G7 Becton Dickinson Immunocytometry, Mountain View,
Calif.
HD37 Dako, Carpinteria, CA
CD20 MEM97 Exbio, Praha, Czech Republic
L27 Becton Dickinson Immunocytometry, Mountain View,
Calif.
CD21 B-Ly4 Pharmingen, San Diego, CA
CD22 HIB22 Pharmingen, San Diego, CA
CD24 32D12 Dr. Steinar Funderud, Institute for Cancer
Research, Dept. of Immunology, Oslo, Norway
ALB9 IMMUNOTECH, Marseille, France
CD25 3G10 Caltaq, Burlingame, CA
CD27 1A4CD27 IMMUNOTECH, Marseille, France
CD29 Lia1.2 IMMUNOTECH, Marseille, France
CD33 =D3HL60.251 IMMUNOTECH, Marseille, France
CD34 581 IMMUNOTECH, Marseille, France
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CD36 FA6.152 IMMUNOTECH, Marseille, France
IVC7 CLB, Central Laboratory of the Netherlands, Red Cross
Blood
Transfusion Service
CD38 T16 IMMUNOTECH, Marseille, France
CD41 P11.64 Kaplan, 5th International Workshop on Human Leukocyte
Differentiation Antigens
SZ22 IMMUNOTECH, Marseille, France
CD42a Bebl Becton Dickinson Irnmunocytometry, Mountain View,
Calif.
CD45 J33 IMMUNOTECH, Marseille, France
MEM28 Exbio, Praha, Czech Republic
CD45RA 8D2.2 Craig et al. 1994, StemCell Technologies, Vancouver,
Canada
L48 Becton Dickinson Immunocytometry, Mountain View,
Calif.
CD45R0 UCHL1 Dako, Carpinteria, CA
CD56 T199 IMMUNOTECH, Marseille, France
MY31 Becton Dickinson Inununocytometry, Mountain View,
Calif.
CD66e CLB/gran10 CLB, Central Laboratory of the Netherlands, Red
Cross Blood
Transfusion Service
CD66b B13.9 CLB, Central Laboratory of the Netherlands, Red Cross
Blood
Transfusion Service
80H3 IMMUNOTECH, Marseille, France
CD69 L78 BD Biosciences, San Jose, CA
CD71 My29 Zymed Laboratories, San Francisco, CA
CD124 S456C9 IMMUNOTECH, Marseille, France
HLADR IMMU357.12 IMMUNOTECH, Marseille, France
IgAl NiF2 IMMUNOTECH, Marseille, France
IgE = G7-18 Phanningen, San Diego, CA
IgG 8A4 IMMUNOTECH, Marseille, France
TCRecri WT31 BD Biosciences, San Jose, CA
TCR Iminu510 IMMUNOTECH, Marseille, France
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TABLE 2
Immunorosetting
Cocktails of Antibodies for Negative Selection of Human Cells
T Cell Enrichment
Anti-
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
Resting T Cell Enrichment
Anti-
HLA-DR and/or CD25, CD69
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
78 T Cell Enrichment
Anti-
afTCR
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
af3T Cell Enrichment
Anti-
yoTCR
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
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TABLE 2 (Coned)
CD4+ T Cell Enrichment
Anti-
CD8
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
Naive CD4+ T Cell Enrichment
Anti-
CD8
CD45R0 and/or CD29
=
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
Memory CD4+ T Cell Enrichment
Anti-
CD8
CD45RA
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
Resting CD4+ T Cell Enrichment
Anti-
CD8
HLA-DR and/or CD25, CD69
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
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TABLE 2 (Cont'd)
CD4+ aBT Cell Enrichment
Anti-
y8TCR
CD8
CD16 and/or C066b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
TH1 CD4+ T Cell Enrichment
Anti-
CD8
CD124
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
TH2 CD4+ T Cell Enrichment
Anti-
CD8
CCR5
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
CD8+ T Cell Enrichment
Anti-
CD4
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
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TABLE 2 (Cont'd1
Naïve CD8+ T Cell Enrichment
Anti-
CD4
CD45R0 and/or CD29
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
Memory CD8+ T Cell Enrichment
Anti-
CD4
CD45RA
=
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
Resting CD8+ T Cell Enrichment
Anti-
CD4
HLA-DR and/or CD25, CD69, CD27
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
CD8+ afiT Cell Enrichment
Anti-
yBTCR
CD4
CD16 and/or CD66b, CD11b, CD15
CD19 and/or CD20, CD21, CD22, CD24, Ig
CD36 and/or CD14
and optionally anti-CD33, CD56, IgE, CD41
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TABLE 2 (coned)
B Cell Enrichment
Anti-
CD2 and/or CD3, both CD4 and CD8
CD16 and/or CD66b, CD11b, CD15
CD36 and/or CD14
and optionally anti-CD33, CD56, CD41
NK Cell Enrichment
Anti-
CD3
CD66b and/or CD15
CD19 and/or CD20, CD21, CD22, CD24
CD36 and/or CD14
and optionally anti-CD33, CD4, IgE, CD41
Monocyte Enrichment
Anti-
CD2 and/or CD3, CD5
CD19 and/or CD20, CD21, CD22, CD24
CD66b and/or CDI6
and optionally anti-CD8, CD56
Dendritic Cell Enrichment
Anti-
CD3
CD14 =
CD16
CD19
CD34
CD56
CD66b
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TABLE 2 (Coned I
Basophil Enrichment
Anti-
CD2
CD3
CD14
CD15
CD16
CD19
CD24
CD34
CD36
CD56
CD45RA
Progenitor Enrichment
Anti-
CD2 and/or CD3
CD16 and/or CD66b
CD19 and/or CD24
CD14
and optionally anti- CD56, CD10, CD45RA, CD38, CD36, CD33, CD71
Erythroid Progenitor Enrichment
Anti-
CD2 and/or CD3
CD16 and/or CD66b
CD19 and/or CD24
CD14
CD45RA
CD33
CD10
and optionally anti- CD56
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TABLE 2 (Cont'd1
Myeloid Progenitor Enrichment
Anti-
CD2 and/or CD3
CD16 and/or CD66b
CD19 and/or CD24
CD14
CD71
CD10
and optionally anti- CD56
Megakaryocyte Progenitor Enrichment
Anti-
CD2 and/or CD3
CD16 and/or CD66b
CD19 and/or CD24
CD14
CD45RA
CD10
and optionally anti- CD56
Epithelial Tumor Cell Enrichment
Anti-
CD45
CD66b
and optionally CD2, CD3, CD14, CD16, CD19, CD36, CD38, CD56, CD66e
=
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TABLE 3
Antibodies Recognizing Non-Hematopoietic
Antigens Expressed on Epithelial Tumor Cells
Specificity Antibody Antigen Supplier/Developer
Epithelial cell BerEp4 ESA, (Epithelial Specific DAKO
markers Antigen) (also known as HEA)
HEA125 ESA Serotec, Cymbus, Pierce,
RDI, Biodesign
VU-1D9 ESA Cymbus
GPI .4 EMA, (Epithelial Membrane IMMUNOTECH, Marseille,
Antigen)(also known as PEM / France
Episialin, a sialomucin)
VU-4H5 EMA Neomarkers
MC.5 EMA Biogenex, also Biodesign
B24.1 EMA Biomeda
E29 EMA DAKO
H11 EGFR DAKO
RAR9941 epithelial glycoprotein Baxter, Germany
. RAR9948 epithelial glycoprotein Baxter, Germany
Carcinoma CU-I 8 BCA 225 (Breast carcinoma ID Labs
(breast, cervical, associated antigen)
ovarian, lung,
endometrial)
Carcinoma 115D8 Carcinoma associated antigen Biogenex, Biodesign
Adenocarcinomas B72.3 TAG-72 (Tumour associated ID Labs, Biogenex,
Signet
glycoprotein)
= Adenocarcinoma B6.2 Unknown, breast cancer marker
Biogenex
s, mammary &
lung carcinomas
Breast Carcinoma 5E11 unknown, breast carcinoma ST1
6E7 unknown, breast carcinoma ST1
H23A unknown, breast carcinoma STI
CA27.29 MAM-6, mucin Cedariane
SM-3 milk mucin core antigen Cymbus, Biodesign,
Imperial
Cancer Research Fund
DF3 CA 15-3 (breast tumour ID Labs
marker)
552 CA 15-3 Biodesign
695 CA 15-3 Biodesign
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 29 -
RAR9938 c-erb B2 Baxter, Germany
C13B5 c-erb B2 IMMUNOTECH, Marseille,
France, also Biogenex
Lung MOC-1 Small cell lung carcinoma ICN Biomed, also
Biodesign
MOC-21 Small cell lung carcinoma ICN Biomed, also
Biodesign
MOC-31 Small cell lung carcinoma ICN Biomed, also
Biodesign
MOC-32 Small cell lung carcinoma ICN Biomed, also
Biodesign
MOC-52 Small cell lung carcinoma ICN Biomed, also
Biodesign
TFS-4 Small cell lung carcinoma Biodesign
Melanoma NKI/C3 Melanoma associated antigen ICN Biomed, also
Biodesign
NKI/M6 Melanoma associated antigen Biodesign
PAL-MI Melanoma associated antigen ICN Biomed, also
Biodesign
HMB45 Melanoma cells Biodesign
Ovarian tumour 185 CA-I25 (ovarian tumour ICN Biomed, also Biodesign
marker)
OV-632 Ovarian cancinoma marker ICN Biomed, also
Biodesign
Gastro-Intestinal CA 19-9 GI tumour marker ICN Biomed, also Biodesign
Cancer
CA 242 GI cancer BioDesign
Renal Cell RC38 Renal Cell Carcinoma Biodesign
Carcinoma
Ewing's Sarcoma 013 Signet
Ewing's Sarcoma CC49 on human adenocarcinomas Signet
Neuroblastoma UJI3A unknown Hurko and Walsh (1983)
Neurology 33:734
UJ181.4 unknown
UJ223.8 unknown
UJ127.11 unknown
5.1.H11 unknown
390,459 unknown R.C. Seeger, L. A. Children's
Hospital, Calif.
BA-1.2 unknown
HSAN 1.2 unknown Reynolds and Smith (1982)
Hybridomas in Cancer p235
CA 02725132 2010-12-09
WO 00/73794 PCT/CA00/00616
- 30 -
TABLE 4
T Cell Enrichment - Immunorosetting Using Ficoll
Purity mean
SD 4
19
Recovery mean = 46
SD = 17
19
SD = Standard deviation from the mean
Purity = % CD3 cells
Recovery = Recovery of CD3+ cells
=
=
=
SUBSTITUTE SHEET (RULE 26)
CA 02725132 2010-12-09
WO 00/73794 PCT/CA00/00616
- 31 -
TABLE 5
CD8+ T Cell Enrichment - Immunorosetting Using Ficoll
% Purity % Recovery
Cocktail n 1SD 1SD
CD4, CD16, CD19, CD36, 19 76 8 44 19
CD56
1
CD4, CD16, CD19, CD36, 5 81 4 j 45 37*
=
CD56, IgE
SD = Standard Deviation from the mean
Purity = % CD8+ cells
Recovery = (vi, Recovery of CD8+ cells
* n = 4
SUBSTII UTE SUIEET (RULE 26)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 32 -
TABLE 6
CD4+ T Cell Enrichment - Immunorosetting Using Ficoll
% Purity % Recovery
Cocktail n - 1SD 1SD
CDS, CD16, CD19, CD36, 19 89 4 57 22
CD56
CD8, CD16, CD19, CD36, 7 93- 3 461-10*
CD56, IgE
SD = Standard Deviation from the mean
Purity = % CD4+ cells
Recovery = % Recovery of CID4+ cells
* n = 5
SUBSTITUTE SHEET (RULE 26)
CA 02725132 2010-12-09
WO 00/73794 PCT/CA00/00616
- 33 -
TABLE 7
B Cell Enrichment - Immunorosetting Using Ficoll
% Purity % Recovery
Cocktail n 1SD 1SD
CD2, CD3, CD16, CD36, 22 72 15 61 27
CD56
CD2, CD3, CD16, CD36, 5 1 88 7 43 18
CD56, IgE
SD = Standard Deviation from the mean
Purity= CD19+ cells
Recovery = % Recovery of CD19+ cells
SINI3T1TUIT, SHEET (RULE 26)
CA 02725132 2010-12-09
WO 00/73794
ItT/CA00/00616
- 34 -
TABLE 8
NK Cell Enrichment - Inununorosetting Using Ficoll
% Purity % Recovery
Cocktail n 1SD 1SD
CD3, CD4, CD19, CD66b, 15 74 10 44 19
CD36
CD3, CD4, CD19, CD66b, 6 88 4
27 20
CD36, IgE
SD = Standard Deviation from the mean.
Purity = (Y. CD56+ cells
Recovery = (Y. recovery CD56+ cells
SUBSTITUTE SHEET (RULE 26)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 35 -
TABLE 9
Enrichment of CD34+ cells from Whole Cord Blood - Immunorosetting
Using Ficoll
% Purity % Recovery
Cocktail n 1SD 1SD
Progenitor 15 29 9 53 29
Enrichment
Debulking 8 l 5 1 45 20
Purity = % CD34+ cells
Recovery = % recovery CD34+ cells
SD = Standard Deviation from the mean
SUOSTri Uri Ct MEE t (1-1t5. n 25)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 36 -
TABLE 10
Monocyte Enrichment - Inununorosetting Using Ficoll
% Purity % Recovery
Cocktail n 1SD 1SD
CD2, CD3, CD19, CD56, 8 71 7 63 28
CD66b
CD2, CD3, CD19, CD56, 5 76 1.5 65 28
CD66b, CD8
CD2, CD3, CD19, CD56, 6 77 4 58 24
CD66b, IgE
CD2, CD3, CD19, CD56, 4 76 3 64 26
CD66b, IgE, CD8
CD2, CD3, CD19, CD56, 1 76 64
CD66b, CD16
CD2, CD3, CD19, CD56, 1 73 41
CD66b, CD20
Purity = %Purity CD14+ cells
Recovery = %Recovery CD14+ cells
SUBSTrTUTE SUM' (RULE 25)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 37 -
TABLE 11
Antibody Composition of Tumor Enrichment Cocktails
Cocktail = Antibodies in Cocktail
CD45 alone CD45
CD45 and CD66b CD45, CD66b
Extensive cocktail CD45, CD2, CD16, CD19, CD36, CD38, CD66b
SUBSTil UTE SHEFT (run E 75)
CA 02725132 2010-12-09
WO 00/73794 PCT/CA00/011616
- 38 -
TABLE 12
Enrichment of CAMA Breast Cancer Cells from Whole Blood
Starting i 1/103 1/104 I 1/105 1/103
iii /10 1/105 1/103 1/104 1/105
frequency
(CAMA)
% Purity of CAMA Log Enrichment of % Recovery of CAMA
Cells CAMA Cells Cells
Enriched 1
CD45 4 2 5 2
0.5 0.4 1.4 0.3 2.2 0.3 2.3 0.4 10 3 26 7 55 36
alone (n=4)
(n=7) (n=3) (n=4) (n=7) .(n=3) (n=4) (n=5) (n=2)
CD45 and 27 4 3.2 0.6 0.5 0.1 2.4 0.1 2.5 0.1 2.7 0.1 15 2 12t1 22 4
' 66b (n=6) (n=6) (n=5) (n=6) (n=6)
(n=5) (n=6) (n=5) (n=5)
Extensive 65 8 26;1-8 3 1 2.8
0.1 3.2 0.2 3.2 0.3 38 8 49 14 33 7
= Cocktail I (n=9) I (n=9) (n=6)
(n=9) (n=9) (n=6) (n=7) (n=5) (n=5)
SUBSTITUTE SHEET (RULE 20)
= CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 39 -
TABLE 13
T Cell Enrichment - Immunorosetting Using Ficoll
Cocktail with CD14 Cocktail with
CD36
1SD t1SD
Purity 3 80 10 94 5
RecoveryI 3 56 12 42 10
SD = Standard Deviation from the mean
= Purity = %Purity CD3+ cells
Recovery = %Recovery CD3+ cells
SUFISTAUTE SHEET MULE 25)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 40 -
TABLE 14
Immunorosetting Using Hetastarch/Iodixanol Mixture
Cell Type Purity Recovery
Enriched mean SD n mean SD
T cells 95% 3% 3 61% 9% 3
CD4+ cells 89% 5% 2 64% 5% 2
CD8+ cells 80% 8% 2 43% 1% 2
B cells 84% 8% 5 58% 26% 5
NK cells - 80% 15% 4 50% 23% 4
SD = Standard Deviation from the mean
Purity = % Purity desired cell type
(T cells = CD3+ cells, CD4+ cells, CD8+ cells, B cells = CD19+
cells, NK cells = CD56+ cells)
Recovery = % Recovery of desired cells
=
=
SUBSTITUTE SHEET (RULE 25)
CA 02725132 2010-12-09
WO 00/73794 PCT/CA00/00616
- 41 -
TABLE 15
Immunorosetting Using Different Layering Media
B Cell Enrichment
Media Ficoll Percoll Iodixanol
Sample 1 (in triplicate)
Purity 1SE 82 2.9 81 1.4 86 2.7
Recovery 1SE 78 6.0 110 3 104 10
Sample 2 (in triplicate)
Purity 1SE 71 1.2 77 1.5 81 2.4
Recovery 1SE 49 8 78 3 64 1
SE = Standrad Error of the mean
Purity = % Purity of CD19+ cells
Recovery = % Recovery of CD19+ cells
SUBS7TFUTE SHEET (RIR E 28)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 42 -
TABLE 16
Puging of Breast Carcinoma Cells Using lmmunorosetting
Sample 1 Sample 2
Log depletion of 1.4, 1.4 1.1, 1.0
CAMA cells
SUBSTITUTE SHEET (RULE 25)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 43 -
TABLE 17
Removal of Granulocytes from Stored Whole Peripheral Blood
Immunorosetting Ficoll alone
% Granulocytes in 1.1, 1.4, 0.7, 0.4 20.9, 18.0
light density fraction
Immunorosetting = Method outlined in Example 2, with depletion
cocktail containing anti-CD66b
Ficoll alone = Standard Ficoll density separation without
immunorosetting
SUBSTITUTE SIM? (mu 25)
CA 02725132 2010-12-09
WO 00/73794
PCT/CA00/00616
- 44 -
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J. Immunol.
Methods, 117:1-8.
4. deWynter, E.A. et al., 1975, Stem Cells, Vol. 13:524-532.
5. Firat et al., 1988, Bone Marrow Transplantation, Vol. 21:933-938.
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Immunol.,
139:2367-2375.
7. Holliger, P., Prospero, T., Winter, G., 1993, Proc. Natl. Acad. Sci.
USA, 90:6444-
6448.
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9. Karawajew, L., Micheel, B., Behrsing, O., Gaestel, M., 1987, J. Immunol.
Methods
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10. Kostelny, S.A., Cole, M.S., Tso, J.Y., 1992, J. Immunol. 148:1547-1553.
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15. Rheinnecker et al., 1996, J. Immunol. 157:2989-2997.
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