U.S. patent application number 10/265622 was filed with the patent office on 2003-08-07 for novel antibody compositions for preparing enriched dendritic cell preparations.
This patent application is currently assigned to StemCell Technologies Inc.. Invention is credited to Lansdorp, Peter, Thomas, Terry.
Application Number | 20030147886 10/265622 |
Document ID | / |
Family ID | 27375914 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030147886 |
Kind Code |
A1 |
Thomas, Terry ; et
al. |
August 7, 2003 |
Novel antibody compositions for preparing enriched dendritic cell
preparations
Abstract
The present invention relates to antibody composition that are
useful in preparing enriched cell preparations such as human
hematopoietic progenitor cells and stem cells and non-hematopoietic
tumor cells. The invention also relates to kits for carrying out
the processes and to the cell preparations prepared by the
processes.
Inventors: |
Thomas, Terry; (Vancouver,
CA) ; Lansdorp, Peter; (Vancouver, CA) |
Correspondence
Address: |
BERESKIN AND PARR
SCOTIA PLAZA
40 KING STREET WEST-SUITE 4000 BOX 401
TORONTO
ON
M5H 3Y2
CA
|
Assignee: |
StemCell Technologies Inc.
Vancouver
CA
|
Family ID: |
27375914 |
Appl. No.: |
10/265622 |
Filed: |
October 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10265622 |
Oct 8, 2002 |
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09428923 |
Oct 28, 1999 |
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6491918 |
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09428923 |
Oct 28, 1999 |
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09088227 |
Jun 1, 1998 |
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6117985 |
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09088227 |
Jun 1, 1998 |
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08566295 |
Dec 1, 1995 |
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6306575 |
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08566295 |
Dec 1, 1995 |
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08491175 |
Jun 16, 1995 |
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5877299 |
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Current U.S.
Class: |
424/144.1 ;
435/7.21 |
Current CPC
Class: |
C07K 16/2809 20130101;
C12N 5/0087 20130101; A61K 38/00 20130101; C12N 5/0693 20130101;
C07K 16/4283 20130101; C07K 16/289 20130101; C07K 16/2803 20130101;
C12N 5/0647 20130101; C07K 16/2806 20130101; C07K 16/18 20130101;
C07K 16/2896 20130101; C07K 16/283 20130101 |
Class at
Publication: |
424/144.1 ;
435/7.21 |
International
Class: |
G01N 033/567; A61K
039/395 |
Claims
We claim:
54. A process for enriching and recovering dendritic cells from a
sample comprising reacting the sample with an antibody composition
containing antibodies capable of binding to the antigens
glycophorin A, CD3, CD14, CD16, CD19, CD34, CD56 and CD66b under
conditions so that conjugates are formed between the antibodies and
the cells in the sample having the antigens glycophorin A, CD3,
CD14, CD16, CD19, CD34, CD56 and CD66b on their surfaces; removing
the conjugates; and recovering a cell preparation which is enriched
in dendritic cells.
56. A process according to claim 54 wherein the sample is whole
blood.
57. A process according to claim 54, wherein the antibodies in the
antibody composition are monoclonal antibodies.
58. A process according to claim 54, wherein the antibodies in the
antibody composition are labelled with a marker or they are
conjugated to a matrix.
59. A process according to claim 54, wherein the antibodies in the
antibody composition are labelled with biotin or a
fluorochrome.
60. A process according to claim 54, wherein the matrix is magnetic
beads, a panning surface, dense particles for density
centrifugation, an adsorption column, or an adsorption
membrane.
61. A process according to claim 57, wherein each of the monoclonal
antibodies in the antibody composition is incorporated in a
tetrameric antibody complex which comprises a first monoclonal
antibody of a first animal species from the antibody composition,
and a second monoclonal antibody of the first animal species which
is capable of binding to at least one antigen on the surface of a
matrix, which have been conjugated to form a cyclic tetramer with
two monoclonal antibodies of a second animal species directed
against the Fc-fragments of the antibodies of the first animal
species.
62. An antibody composition for enriching for dendritic cells
comprising antibodies specific for glycophorin A, CD3, CD14, CD16,
CD19, CD34, CD56 and CD66b.
63. An antibody composition according to claim 62, wherein the
antibodies are monoclonal antibodies.
64. An antibody composition according to claim 63 wherein the
antibodies are labelled with a marker or they are directly or
indirectly conjugated to a matrix.
65. An antibody composition according to claim 63 wherein the
antibodies are labelled with biotin or a fluorochrome.
66. An antibody composition according to claim 64 wherein the
matrix is magnetic beads, a panning surface, dense particles for
density centrifugation, an adsorption column, or an adsorption
membrane.
67. An antibody composition according to claim 63 wherein the
antibodies are joined to a cytotoxic agent.
68. An antibody composition according to claim 63 wherein each of
the monoclonal antibodies is incorporated in a tetrameric antibody
complex which comprises a first monoclonal antibody of a first
animal species from the antibody composition, and a second
monoclonal antibody of the first animal species which is capable of
binding to at least one antigen on the surface of a matrix, which
have been conjugated to form a cyclic tetramer with two monoclonal
antibodies of a second animal species directed against the
Fc-fragments of the antibodies of the first animal species.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 09/088,227 filed on Jun. 1, 1998 which is a
continuation-in-part of U.S. patent application Ser. No.
08/566,295, filed Dec. 1, 1995, which is a continuation-in-part of
U.S. patent application Ser. No. 08/491,175, filed Jun. 16, 1995,
both of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel antibody
compositions, and processes and kits for preparing enriched cell
preparations, such as cell preparations enriched in human
hematopoietic progenitor cells or stem cells or non-hematopoietic
tumor cells.
BACKGROUND OF THE INVENTION
[0003] Blood cells have a relatively short life span and need to be
replenished throughout life. In adults, blood cell formation or
hematopoiesis takes place in the bone marrow, but blood-forming
stem cells can also be found in peripheral blood. Hematopoietic
cells represent a hierarchy of proliferating and differentiating
cells. The most abundant are the differentiating or lineage
committed cells. These cells have limited or no proliferative
capacity and represent specialized end cells that are found in
blood, and their immediate precursors.
[0004] The immediate precursors of the differentiating cells are
the progenitor cells. Most of these cells are restricted to
differentiate along a single lineage but they may have quite
extensive proliferative capacity. Progenitor cells appear
morphologically as blast cells, and they typically do not have
specific features of the hematopoietic lineage to which they are
committed.
[0005] Progenitor cells are derived from stem cells. Stem cells
have been historically defined as cells capable of long term
hematopoietic repopulation. This implies their ability to
self-renew as well as to generate daughter cells of any of the
hematopoietic lineages. The presence of stem and progenitor cells
may be detected by their ability to produce colony-forming cells in
culture and repopulate xenogeneic hosts such fetal sheep (Zanjani
et al., 1994 J. Clin. Invest. Vol. 89, p. 1178-1188) and
immuno-deficient mice (Dick et al., 1991 Immunological Reviews,
Vol. 124:25-43). They may also be detected by screening for the
CD34 antigen which is a positive marker for early hematopoietic
cells including colony forming cells and stem cells. At present,
the long term culture initiating cell (LTCIC) assay appears to be
the best way to detect stem cells, or at least the most primitive
progenitor cells, using tissue culture methodologies.
[0006] There is a continued interest in developing stem cell
purification techniques. Pure populations of stem cells will
facilitate studies of hematopoiesis. Transplantation of
hematopoietic cells from peripheral blood and/or bone marrow is
also 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.
[0007] Hematopoietic 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 bone marrow and peripheral blood
using monoclonal antibodies. They differ in whether it is the
desired or undesired cells which are distinguished/labeled with the
antibody(s).
[0008] In positive selection techniques the desired cells are
labeled with antibodies and removed from the remaining
unlabeled/unwanted cells. In negative selection, the unwanted cells
are labeled and removed. Antibody/complement treatment and the use
of immunotoxins are negative selection techniques, but FACS sorting
and most batch wise immunoadsorption 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.
[0009] Current positive selection techniques for the purification
of hematopoietic stem cells target and isolate cells which express
CD34 (approximately 1-2% of normal bone marrow) (Civin, C. l.,
Trischmann, T. M., Fackler, M. J., Bernstein, I. D., Buhring, H.
J., Campos, L. et al. 1989 Report on the CD34 cluster workshop, In:
Leucocyte typing IV, White Cell Differentiation Antigens. Knapp,
W., Dorken, B., Gilks, W. R., Reiber, E P., Schmidt, R. E., Stein,
H., and Kr. von den Borne, AE.G Eds., Oxford University Press.
Oxford, pp.818). Thus, the potential enrichment of hematopoietic
stem cells using this marker alone is approximately 50 fold.
Available techniques typically recover 30-70% of the CD34+cells in
the start suspension and produce an enriched suspension which is
50-90% CD34.sup.+ (Firat et al., 1988, Bone Marrow Transplantation,
Vol. 21:933-938; deWynter, E. A. et al., 1975, Stem Cells, Vol.
13:524-532; Shpall, E. J., et al. 1994, J. of Clinical Oncology
12:28-36; Thomas, T. E., 1994, Cancer Research, Therapy and Control
4(2): 119-128). The positive selection procedures suffer from many
disadvantages including the presence of materials such as
antibodies and/or magnetic beads on the CD34.sup.+ cells, and
damage to the cells resulting from the removal of these materials.
Also to be considered is the recent evidence that some long term
repopulating cells are CD34.sup.- (negative) (Zanjani et al., 1998,
Exp. Hematol., Vol. 26:353-360) and methods that isolate CD34.sup.+
will not capture these cells.
[0010] Negative selection has been used to remove minor populations
of cells from clinical grafts. These cells are either T-cells or
tumor cells that pose a risk to the transplant recipient. The
efficiency of these purges varies with the technique and depends on
the type and number of antibodies used. Typically, the end product
is very similar to the start suspension, missing only the tumor
cells or T-cells.
[0011] Transplants of purified stem cells without differentiated or
lineage committed cells will give short and long-term hematopoietic
support (Shpall, E. J., et al. 1994, J. of Clinical Oncology
12:28-36). Since differentiated cells make up a vast majority of
the cells in bone marrow and blood, depletion of these cells
produces a much smaller cell suspension. The number of cells in the
final product and the degree of enrichment of progenitor/stem cells
will depend on the efficiency of the antibody targeting and the
removal of labeled cells.
[0012] There are several studies that enrich for hematopoietic stem
cells by depleting lineage committed cells but all require a number
of positive or negative selection steps to achieve the desired
degree of enrichment (50 fold). Early studies required prior
density separation and extensive incubations to remove adherent
cells (Linch, D. C, and Nathan, D. G. 1984, Nature 312 20/27:
775-777; Sieff, C. A., et al., 1985, Science 230: 1171-1173;
Kannourakis, G. and Bol, S., 1987 Exp. Hematol 15:1103-1108.). More
recent techniques are no less cumbersome; involving density
separation steps followed by two partial lineage depletions
(Winslow, J. M., et al., 1994, Bone Marrow Transplantation
14:265-271) or a partial lineage depletion using panning or FACS
followed finally by positive selection using FACS (Carlo-Stella et
al. 1994, Blood 84, 10 supple.:104a; Reading, C., et al. (1994),
Blood 84, 10 supple.:399a). Most of these methods for lineage
depletion lack effective antibody combinations against myeloid
cells, erythrocytes and/or B-cells.
[0013] U.S. Pat. No. 5,087,570 describes a process for preparing a
hematopoietic cell composition using a combination of positive and
negative selection. The process relies on the use of antibody to
the Sca-1 antigen which is associated with murine clonogenic bone
marrow precursors of thymocytes and progeny T-cells. The Sca-1
antibody is not useful in isolating human hematopoietic cells.
[0014] 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 (Vaughan et al., 1990, Proc. Am. Soc. Clin.
Oncol. 9:9). Our ability to detect such metastatic cells is limited
by the effectiveness of tissue or fluid sampling and the
sensitivity of tumor detection methods. From a research point of
view, it is also very difficult to study such rare cells and
determine the biological changes which enable spread of disease.
Metastatic epithelial tumor cells disseminate to distant sites such
as bone marrow and lymph nodes. Bone marrow has become an important
indicator organ for the spread of epithelial cells because of its
easy accessibility and the lack of normal epithelial cells making
identification of tumor cells less difficult. The recent trend in
autologous transplantation away from the use of bone marrow grafts
to cytokine mobilized peripheral blood has raised the question of
how often peripheral blood is contaminated with micrometastatic
tumor cells. Epithelial tumor contamination in peripheral blood is
less frequent than in bone marrow (Ross et al., 1993, Blood,
82(9):2605-2610) but cytokine mobilization may also "mobilize"
tumor cells (Brugger et al., 1994, Blood, 83(3):636-640). Both
cancer research and patient therapy could benefit from method of
enriching epithelial tumor cells from blood, bone marrow and
peritoneal and pleural effusions.
[0015] The two most poplar methods in research laboratories for the
detection of rare epithelial tumor cells are immuno-cytochemical
staining (ICC) and polyermase chain reaction (PCR). PCR detects
specific DNA or RNA sequences. ICC methods rely on antibodies to
epithelial-specific cytoskeleton and membrane antigens to stain
tumor cells. ICC is more widely used clinically and established
laboratories with experienced staff are consistently reporting
sensitivities of one tumor cell is 10.sup.5 bone marrow cells
(Pantel, 1996, J. of Hematotherapy, 5:359-367). An enrichment of
100 fold or 2 log could increase this sensitivity to one in
10.sup.7 cells.
[0016] There are two approaches to enriching epithelial tumor cells
from a suspension of non-epithelial cells such as bone marrow or
blood. One can either target the tumor cells for recovery using an
epithelial or tumor specific antibodies (positive selection) or
target all the non-epithelial (in this case hematopoietic cells)
for depletion (negative selection). The problems with the first
approach, positive selection, is that the recovered tumor cells are
covered with antibodies and the sites commonly used for
immunocytochemical detection are blocked. It is also difficult to
positively select cells from samples that have been stored or
previously frozen. The non-specific binding of antibodies to cells
or of cells to the separation matrix are too high. Negative
selection, on the other hand, can deal with clumpy or previously
frozen cell suspensions (Thomas et al., 1998, Methods in
Enzymology: Signalling Pathways and Gene Regulation in
Hematopoietic Cell Growth and Differentiation "Purification of
Hematopoietic Stem cells for Further Biological Study", Academic
Press) as the recovered cells have not been labelled with antibody.
Both currently available epithelial tumor cells enrichment methods
are positive selections using cytokeratin specific antibodies or
antibodies to Human Epithelial Antigen (HEA) (Miltenyi Biotec Inc.
Auburn Calif.; and Dynal, Skoyen Norway). A negative selection
technique that employs antibodies to CD45 has also been reported
but enrichments are only 1-2 log and vary with cell source. Van
Vlasselaer (U.S. Pat. No. 5,648,223) teaches a procedure for
enriching tumor cells in whole blood using cell-trap centrifugation
to enrich tumor cells in circulating bodily fluids, by separation
based on density. However, the methods taught by Van Vlasselaer
require the construction and operation of a cell trap centrifuge
tube calibrated to specific gradients of density, osmolality and
pH.
[0017] In order to successfully utilize circulating bodily fluids
for cancer diagnosis, improved methods of enriching the small
number of circulating tumor cells are required.
SUMMARY OF THE INVENTION
[0018] The present inventors have developed antibody compositions
for use in preparing cell preparations enriched for certain cell
types such as human hematopoietic stem cells and progenitor cells
as well as non-hematopoietic tumor cells found in blood, bone
marrow, pleural and peritoneal effusions.
[0019] To enrich for hematopoietic stem cells and progenitor cells,
the antibodies in the antibody composition are specific for
selected markers associated with lineage committed or
differentiated cells thereby allowing them to be removed from the
cell preparation. In particular, the present inventors have found
using a negative selection technique that an antibody composition
containing antibodies specific for glycophorin A, CD3, CD24, CD16,
and CD14 gives a cell preparation highly enriched for human
hematopoietic and progenitor cells. Preferably, the composition
additionally includes antibodies to CD56, CD2, CD19, CD66e and/or
CD66b. To enrich for early progenitor and stem cells (CD34.sup.+,
CD38.sup.- cells), the antibody composition also includes
antibodies to CD45RA, CD36 and CD38.
[0020] The present inventors have shown that the use of the
antibody composition of the present invention in a negative
selection technique, to prepare a cell preparation which is
enriched for hematopoietic stem cells and progenitor cells offers
many advantages over conventional techniques. The antibody
composition applied in one step to a sample of peripheral blood,
bone marrow, cord blood or frozen bone marrow, results in a greater
than 50% recovery of human hematopoietic progenitor/stem cells with
approximately a 3 log depletion of differentiated cells.
[0021] In addition to enriching for hematopoietic progenitor and
stem cells, the above-described antibody compositions can be used
to deplete tumor cells derived from hematopoietic cells such as
B-cell lymphomas or T-cell leukemias. Accordingly, the present
invention also provides an antibody composition to enrich for
hematopoietic stem cells and progenitor cells and to remove
hematopoietic tumor cells. The composition comprises antibodies
specific for glycophorin A, CD3, CD24, CD16, and CD14. The
composition preferably also includes antibodies to CD2, CD56, CD19,
CD66e and/or CD66b.
[0022] The present invention also includes an antibody composition
to enrich for hematopoietic stem cells and progenitor cells and to
remove non-hematopoietic tumor cells. In such an embodiment, the
composition also 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. Accordingly, the present invention provides an
antibody composition to enrich for hematopoietic stem cells and
progenitor cells and to remove tumor cells comprising antibodies
specific for glycophorin A, CD3, CD24, CD 16, CD14 and an antigen
present on the tumor cells. The antigens on the tumor cells is
preferably a non-hematopoietic antigen expressed on the tumor
cells.
[0023] The present inventors have shown that the purging antibody
composition applied in one step to a sample of peripheral blood,
bone marrow, or frozen bone marrow containing tumor cells, results
in a greater than 50% recovery of human hematopoietic
progenitor/stem cells with approximately a 3-5 log depletion of
tumor cells.
[0024] The high level of enrichment obtained using the antibody
compositions of the invention, does not require additional
enrichment or tumor purging steps, which would result in loss of,
or damage to, progenitor and stem cells. The recovery of CD34.sup.+
cells, CD34.sup.+CD38.sup.- cells, colony forming cells, and LTCIC,
is also much higher than with conventional multistep
techniques.
[0025] The present inventors have also developed an antibody
composition for use in preparing cell preparations enriched for
non-hematopoietic tumor cells, in particular metastatic tumor
cells. The composition is useful in the detection of
non-hematopoietic tumor cells from blood and bone marrow of
patients to aid in the detection of metastatic disease. The
tumor-enriching antibody composition contains antibodies specific
for selected markers associated with hematopoietic cells. In
particular, the present inventors have found using a negative
selection technique that an antibody composition containing
antibodies specific for glycophorin A, CD2, CD14, CD16, CD38, CD45
and CD66b, and optionally CD3, CD36, CD56, and/or CD66e, gives a
cell preparation highly enriched for non-hematopoietic tumor cells.
The present inventors have shown that the tumor enriching antibody
compositions applied in one step to a sample of peripheral blood,
frozen peripheral blood, bone marrow or pleural or peritoneal
effusions containing tumor cells results at least a 2 log
enrichment (and typically greater than 3 log enrichment), of the
tumor cells.
[0026] The enrichment and recovery of human hematopoietic
progenitor and stem cells as well as non-hematopoietic tumor cells
using the antibody compositions of the invention in a negative
selection technique has many advantages over conventional positive
selection techniques. As mentioned above, highly enriched cell
preparations can be obtained using a single step. The cells
obtained using the antibody composition of the invention are not
labeled or coated with antibodies or modified making them highly
suitable for many uses. For example, the isolated hematopoietic
stem cells and progenitor cells can be used in transplantation and
other therapeutic uses. The isolated metastatic tumor cells can be
used to detect metastatic disease in blood and bone marrow as well
as pleural and peritoneal effusions.
[0027] The present invention also relates to a negative selection
process for enriching and recovering human hematopoietic progenitor
cells and stem cells in a sample containing human hematopoietic
differentiated, progenitor, and stem cells comprising (a) reacting
the sample with an antibody composition containing antibodies
capable of binding to the antigens glycophorin A, CD3 CD24, CD16,
and CD14, and optionally CD2, CD56, CD19, CD66e and/or CD66b under
conditions permitting the formation of conjugates between the
antibodies and cells in the sample having the antigens glycophorin
A, CD3 CD24, CD16, and CD14, and optionally CD2, CD56, CD19, CD66e
and/or CD66b on their surfaces; (b) removing the conjugates; and
(c) recovering a cell preparation which is enriched in human
hematopoietic progenitor cells and stem cells.
[0028] The present invention further provides a negative selection
process for enriching and recovering normal human hematopoietic
progenitor cells and stem cells and depleting hematopoietic tumor
cells in a sample containing human hematopoietic differentiated,
progenitor, and stem cells, and hematopoietic tumor cells
comprising (a) reacting the sample with an antibody composition
containing antibodies capable of binding to the antigens
glycophorin A, CD3, CD24, CD16, CD14, and optionally CD2, CD56,
CD19, CD66e and/or CD66b, under conditions so that conjugates are
formed between the antibodies and the cells in the sample having
the antigens glycophorin A, CD3 CD24, CD16, and CD14, and
optionally CD2, CD56, CD19, CD66e and/or CD66b; (b) removing the
conjugates; and (c) recovering a cell preparation which is enriched
in normal human hematopoietic progenitor cells and stem cells and
depleted in hematopoietic tumor cells.
[0029] The present invention further provides a process for
enriching and recovering human hematopoietic stem cells and
progenitor cells and depleting tumor cells in a sample containing
differentiated cells, progenitor cells, stem cells and tumor cells,
comprising (a) reacting the sample with an antibody composition
containing antibodies capable of binding to the antigens
glycophorin A, CD3 CD24, CD16, and CD14, and an antigen present on
the tumor cells and optionally CD2, CD56, CD19, CD66e and/or CD66b
under conditions permitting the formation of conjugates between the
antibodies and cells in the sample having the antigens glycophorin
A, CD3 CD24, CD16, and CD14, and an antigen present on the tumor
cells and optionally CD2, CD56, CD19, CD66e and/or CD66b on their
surfaces; (b) removing the conjugates; and (c) recovering a cell
preparation which is enriched in human hematopoietic progenitor
cells and stem cells and depleted in tumor cells.
[0030] The present invention also contemplates a negative selection
process for enriching for non-hematopoietic metastatic tumor cells
in a sample containing the tumor cells and hematopoietic cells
comprising (a) reacting the sample with an antibody composition
comprising antibodies specific for glycophorin A, CD2, CD14, CD16,
CD38, CD45 and CD66b, and optionally CD3, CD36, CD56 and/or CD66e
under conditions so that conjugates are formed between the
antibodies and hematopoietic cells in the sample expressing the
antigens glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b, and
optionally CD3, CD36, CD56 and/or CD66e; (b) removing the
conjugates; and (c) recovering a cell preparation enriched in the
tumor cells.
[0031] The present invention also relates to a kit useful in
preparing a cell preparation enriched in human hematopoietic
progenitor and stem cells comprising antibodies specific for
glycophorin A, CD3, CD24, CD16, and CD14, and instructions for
preparing a cell preparation enriched in hematopoietic progenitor
and stem cells.
[0032] The present invention further includes a kit useful in
preparing a cell preparation enriched in hematopoietic stem cells
and progenitor cells and depleted in hematopoietic tumor cells
comprising antibodies specific for glycophorin A, CD3, CD24, CD16,
and CD14 and instructions for preparing a cell preparation enriched
in hematopoietic stem cells and progenitor cells and depleted in
hematopoietic tumor cells.
[0033] The present invention also includes a kit useful in
preparing a cell preparation enriched in hematopoietic stem cells
and progenitor cells and depleted in tumor cells comprising
antibodies specific for glycophorin A, CD3, CD24, CD16, CD14, and
an antigen present on the tumor cells and instructions for
preparing a cell preparation enriched in hematopoietic stem cells
and progenitor cells and depleted in tumor cells.
[0034] The present invention also relates to a kit useful in
preparing a cell preparation enriched in non-hematopoietic tumor
cells from blood, bone marrow, pleural or peritoneal effusions,
comprising antibodies specific for glycophorin A, CD2, CD14, CD16,
CD38, CD45 and CD66b, and instructions for preparing a cell
preparation enriched in non-hematopoietic tumor cells.
[0035] The invention further relates to cell preparations obtained
in accordance with the processes of the invention. The invention
still further contemplates a method of using the antibody
compositions of the invention in negative selection methods to
recover a cell preparation which is enriched in human hematopoietic
progenitor and stem cells or non-hematopoietic tumor cells.
[0036] These and other aspects of the present invention will become
evident upon reference to the following detailed description and
attached drawings. In addition, reference is made herein to various
publications, which are hereby incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] The invention will now be described with reference to the
accompanying drawings, in which:
[0038] FIG. 1 is a schematic representation of magnetic cell
labeling using tetrameric antibody complexes and colloidal dextran
iron; and
[0039] FIG. 2A shows a Fluorescence Activated Cell Sorting (FACS)
histogram of mobilized peripheral blood before progenitor
enrichment using the progenitor enrichment composition.
[0040] FIG. 2B shows a Fluorescence Activated Cell Sorting (FACS)
histogram of mobilized peripheral blood after progenitor enrichment
using the progenitor enrichment composition.
[0041] FIG. 3A shows a Fluorescence Activated Cell Sorting (FACS)
profile of mobilized peripheral blood before enrichment using the
primitive progenitor enrichment composition.
[0042] FIG. 3B shows a Fluorescence Activated Cell Sorting (FACS)
profile of mobilized peripheral blood after enrichment using the
primitive progenitor enrichment composition.
[0043] FIG. 4A shows a Fluorescence Activated Cell Sorting (FACS)
profile of peripheral blood seeded with CAMA Breast carcinoma cell
line before enrichment using the tumor enrichment composition.
[0044] FIG. 4B shows a Fluorescence Activated Cell Sorting (FACS)
profile of peripheral blood seeded with CAMA Breast carcinoma cell
line after enrichment using the tumor enrichment composition.
[0045] FIG. 5A shows a Fluorescence Activated Cell Sorting (FACS)
profile of peripheral blood seeded with pleural effusion cells
before enrichment using the tumor enrichment composition.
[0046] FIG. 5B shows a Fluorescence Activated Cell Sorting (FACS)
profile of peripheral blood seeded with pleural effusion cells
after enrichment using the tumor enrichment composition.
DETAILED DESCRIPTION OF THE INVENTION
[0047] I. Hematopoietic Cell Types and Tumor Cells
[0048] The term "differentiated cells" used herein refers to human
hematopoietic cells which have limited or no proliferative
capacity. Differentiated cells represent specialized end cells that
are found in blood, and their immediate precursors.
[0049] The term "progenitor cells" used herein refers to cells
which are the immediate precursors of the differentiating cells.
Most of the progenitor cells differentiate along a single lineage
but they may have quite extensive proliferative capacity.
Progenitor cells appear morphologically as blast cells, and they
typically do not have specific features of the hematopoietic
lineage to which they are committed.
[0050] The term "stem cells" used herein refers to the cells from
which progenitor cells are derived. Stem cells are defined by their
ability to self-renew as well as to generate daughter cells of any
of the hematopoietic lineages. Stem cells with long term
hematopoietic reconstituting ability can be distinguished by a
number of physical and biological properties from differentiated
cells and progenitor cells (Hodgson, G. S. & Bradley, T. R.,
Nature, Vol. 281, pp. 381-382; Visser et al., 1984, J. Exp. Med.,
Vol. 59, pp. 1576-1590; Spangrude et al., 1988, Science, Vol. 241,
pp. 58-62; Szilvassy et al., 1989, Blood, Vol. 74, pp. 930-939;
Ploemacher, R. E. & Brons, R. H. C., 1989, Exp. Hematol., Vol.
17, pp. 263-266).
[0051] The presence of stem cells and progenitor cells in a cell
preparation may be detected by their ability to produce
colony-forming cells in culture or to repopulate xeonogenic hosts
such as immunodeficient mice. They may also be detected by
screening for the CD34 antigen which is a positive marker for early
hematopoietic cells including colony forming cells and stem cells.
Primitive hematopoietic stem cells with long term hematopoietic
reconstituting ability can be identified by determining the number
of clonogenic cells present after 5 to 8 weeks in long term
cultures (Sutherland et al., 1986, Blood, Vol. 74, p. 1563;
Udomsakdi et al., 1991, Exp. Hematol., Vol. 19, p. 338; and,
Sutherland et al., 1990, Proc. Natl. Acad. Sci., Vol. 87, p.
3584).
[0052] Tumor cells which may be removed from a sample using the
antibody compositions and processes described herein include tumor
cells which have non-hematopoietic antigens or markers expressed on
their surfaces i.e. antigens that distinguish the tumor cells from
hematopoietic progenitor cells and stem cells. For example,
specific markers have been found to be expressed on tumor cells
such as breast and lung carcinoma, and neuroblastoma. Table 4 lists
specific examples of antibodies which recognize non-hematopoietic
antigens expressed on tumor cells.
[0053] Some metastatic tumor cells express hematopoietic lineage
markers or antigens, for example, tumor cells from B-lymphomas,
multiple myeloma, some chronic lymphocytic leukemias (CLL), and
some acute lymphocytic leukemias (ALL) express B-cell markers such
as CD22, CD20, CD29, and T cells from ALL and CLL express T-cell
markers, and antibodies to these antigens may be included in the
antibody compositions of the invention to remove tumor cells
expressing the hematopoietic lineage antigens.
[0054] Tumor cells which may be enriched in a sample using the
antibody compositions and processes described herein include
non-hematopoietic tumor cells which do not express hematopoietic
lineage markers. Non-hematopoietic tumors include epithelial
cancers of the bronchi, mammary ducts, gastrointestinal tract,
reproductive system and urogenital tract such as carcinomas of the
lung, breast, colon, prostate, bladder, ovary, endometrium, cervix,
pancreas, oesophagus, small bowel, rectum, uterus, stomach, larynx,
skin and vagina.
[0055] II. Antibody Compositions
[0056] As hereinbefore mentioned, the invention relates to an
antibody compositions for preparing enriched cell preparations. In
one aspect, the antibody composition is for enriching human
hematopoietic progenitors and stem cells and comprises antibodies
specific for the antigens glycophorin A, CD3, CD24, CD16, and CD14,
which are present on the surface of human differentiated cells. In
a preferred embodiment, the antibody composition further includes
antibodies to CD2, CD56, CD19, CD66e and/or CD66b. The composition
may also include antibodies to CD45RA, CD38, and/or CD36.
[0057] The antibody composition for enriching for human
hematopoietic progenitor and stem cells may be generally referred
to as the "progenitor enrichment composition" or the "progenitor
enrichment cocktail". One skilled in the art will appreciate that
in addition to the antibodies listed above, the progenitor
enrichment cocktail may additionally include other antibodies that
are specific for antigens on the surface of differentiated cells
including those listed in Table 2. The selection of the antibodies
can depend on many factors including the nature of the sample to be
enriched. In an embodiment of the invention, an antibody
composition is provided for enriching and recovering human
hematopoietic progenitor and stem cells from fresh bone marrow
consisting of antibodies specific for glycophorin A, CD3, CD24,
CD16, CD14, CD66e and CD66b. In a second embodiment, an antibody
composition is provided for enriching and recovering human
hematopoietic progenitor and stem cells from previously frozen bone
marrow consisting of antibodies specific for glycophorin A, CD3,
CD24, CD16, and CD14. In a further embodiment of the invention, an
antibody composition is provided for enriching and recovering human
hematopoietic progenitor and stem cells from peripheral or cord
blood consisting of antibodies specific for glycophorin A, CD3,
CD24, CD16, CD14, CD66e, CD66b, CD56, CD2 and CD19.
[0058] Pluripotent stem cells and committed progenitors express
CD34, and this CD34 compartment can be subdivided using antibodies
to a variety of cell surface markers. Stem cells co-purify in a
population of CD34.sup.+ cells which lack or have low expression of
certain lineage markers (CD38, CD33, CD45RA, CD71, CD36 and HLA-DR)
(Craig et al. 1994, British Journal of Haematology, 88:24-30;
Lansdorp, P. A I. and Dragowska, W. 1992 J. Exp. Med.
175:1501-1509; Sutherland, H, J., et al. 1989 Blood 74.1563-1570).
Antibodies recognizing these antigens can be included in the
antibody composition to further enrich for stem cells, while losing
some of the committed mature CD34.sup.+ cells. Preferably,
anti-CD45RA, anti-CD38 and anti-CD36 are included in the antibody
composition. Accordingly, in another embodiment the present
invention provides an antibody composition for enriching for early
progenitor cells comprising antibodies specific for glycophorin A,
CD3, CD24, CD16, CD14, CD2, CD56, CD19, CD66b, CD45RA, CD36 and
CD38.
[0059] In another aspect, the present invention also relates to an
antibody composition for enriching and recovering human
hematopoietic progenitor and stem cells and depleting hematopoietic
tumor cells comprising antibodies specific for glycophorin A, CD3,
CD24, CD16 and CD14. In a preferred embodiment the antibody
composition further includes antibodies to CD2, CD56, CD19, CD66e
and/or CD66b.
[0060] The present invention also includes an antibody composition
for enriching and recovering hematopoietic stem cells and
progenitor cells and depleting non-hematopoietic tumor cells. In
such an embodiment, the composition also 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 tumor antigens may be obtained from commercial sources or
prepared using techniques known in the art. Preferably, the
antibodies specific for non-hematopoietic antigens are specific for
antigens expressed on breast and lung carcinoma and neuroblastoma
cells, for example a shown in Table 4.
[0061] In a further aspect, the invention also includes an antibody
composition for enriching and recovering non-hematopoietic tumor
cells from blood, bone marrow, pleural and peritoneal effusions
comprising antibodies specific for glycophorin A, CD2, CD14, CD16,
CD38, CD45 and CD66b, and optionally CD3, CD36, CD56 and/or
CD66e.
[0062] 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').sub.2) and
chimeric antibodies. Antibodies are understood to be reactive
against a selected antigen on the surface of a differentiated cell
or tumor cell if they bind with an appropriate affinity
(association constant), e.g. greater than or equal to 10.sup.7
M.sup.-1.
[0063] Polyclonal antibodies against selected antigens on the
surface of differentiated cells or tumor cells may be readily
generated by one of ordinary skill in the art from a variety of
warm-blooded animals such as horses, cows, various fowl, rabbits,
mice, hamsters, or rats. For example, a mammal, (e.g., a mouse,
hamster, or rabbit) can be immunized with an immunogenic form of an
antigen which elicits an antibody response in the mammal.
Techniques for conferring immunogenicity on an antigen include
conjugation to carriers or other techniques well known in the art.
For example, the antigen can be administered in the presence of
adjuvant. The progress of immunization can be monitored by
detection of antibody titers in plasma or serum. Following
immunization, antisera can be obtained and polyclonal antibodies
isolated from the sera.
[0064] Monoclonal antibodies are preferably used in the antibody
compositions of the invention. Monoclonal antibodies specific for
selected antigens on the surface of differentiated cells or tumor
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. Pat. No. RE 32,011, U.S. Pat. Nos.
4,902,614, 4,543,439, and 4,411,993 which are incorporated herein
by reference; see also Monoclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Plenum Press, Kennett, McKearn,
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 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.
[0065] The term "antibody" as used herein is intended to include
antibody fragments which are specifically reactive with specific
antigens on the surface of differentiated cells or tumor cells.
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').sub.2 fragments can
be generated by treating antibody with pepsin. The resulting
F(ab').sub.2 fragment can be treated to reduce disulfide bridges to
produce Fab' fragments.
[0066] 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 immunoglobulin 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. Pat. No. 4,816,567; Boss et al., U.S.
Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication
EP171496; European Patent Publication 0173494, United Kingdom
patent GB 2177096B.
[0067] Antibodies against selected antigens on the surface of
differentiated cells or tumor cells may also be obtained from
commercial sources as illustrated in Tables 2 and 4.
[0068] Antibodies may be selected for use in the antibody
compositions of the invention based on their ability to deplete
targeted differentiated cells and/or tumor cells and recover
non-targeted cells (i.e. normal progenitor and stem cells, or
specific differentiated cells) in magnetic cell separations as more
particularly described herein, and in U.S. Pat. No. 5,514,340,
which is incorporated in its entirety herein by reference. In
general, an antibody is selected that gives greater than 3 log
depletion of differentiated cells or tumor cells, with greater than
75% recovery of CD34.sup.+ cells (bone marrow, mobilized blood and
cord blood) or non-targeted lymphocytes (steady state blood), in
test magnetic cell separations as described herein.
[0069] The anti-glycophorin A antibodies contained in the antibody
composition of the invention are used to label erythrocytes.
Examples of monoclonal antibodies specific for glycophorin A are
2B7.1 (StemCell Technologies), 10F7MN (U.S. Pat. No. 4,752,582,
Cell lines: ATCC accession numbers HB-8162), and D2.10 (Immunotech,
Marseille, France). The concentration of antiglycophorin A
antibodies used in the antibody composition are generally less than
the concentration that will cause agglutination (i.e. 3-10
.mu.g/ml). Preferably the concentration of antiglycophorin A
antibodies used in the antibody composition is between about 0.5 to
5 .mu.g/ml, preferably 1 to 2 .mu.g/ml.
[0070] Monoclonal antibodies against CD24, CD3, CD19, CD20, CD22,
CD29, CD56, CD2 in the antibody composition of the invention are
used to label B and T lymphocytes and NK cells. Examples of
monoclonal antibodies specific for CD24, CD3, CD19, CD20, CD22,
CD56, and CD2, are 32D12 (Dr. Steinar Funderud, Institute for
Cancer Research, Dept. of Immunology, Oslo, Norway,) and ALB9
(Immunotech, Marseille, France); UCHT1 (Immunotech, Marseille,
France) and SK7 (Becton Dickinson, Mountain View, Calif.); J4.119
(Immunotech, Marseille, France) and Leu-12 (Becton Dickinson,
Mountain View, Calif.); MEM97 (Dr. Horejsi, Institute of Molecular
Genetics Academy of Sciences of the Czech Republic, Praha, Czech
Republic, or Cedarlane Laboratories, Homby, Ontario, Canada) and
Leu-16 (Becton Dickinson, Mountain View, Calif.); SJ10.1H11
(Immunotech, Marseille, France); T199 (Immunotech, Marseille,
France); and 6F10.3 (Immunotech, Marseille, France), respectively.
The concentration of each of the monoclonal antibodies against
CD24, CD3, CD19, CD20, CD56, CD2 contained in the antibody
composition is between about 0.5 to 5 .mu.g/ml, preferably 2 to 3
.mu.g/ml.
[0071] Monoclonal antibodies against CD14, CD16, CD66e and CD66b in
the antibody compositions of the invention are used to label
monocytes and granulocytes. Examples of monoclonal antibodies
specific for CD14, CD16, CD66e and CD66b, are MEM15 and MEM18 (Dr.
Vaclav Horejsi, Institute of Molecular Genetics Academy of Sciences
of the Czech Republic, Praha, Czech Republic; Cedarlane
Laboratories, Homby, Ontario, Canada); MEM154 (Dr. Vaclav Horejsi,
Institute of Molecular Genetics Academy of Sciences of the Czech
Republic, Praha, Czech Republic; Cedarlane Laboratories, Homby,
Ontario, Canada), Leu-lla (Becton Dickinson, Mountain View,
Calif.), and 3G8 (Immunotech, Marseille, France); CLB/gran10 (CLB,
Central Laboratory of the Netherlands, Red Cross Blood Transfusion
Service); and, B13.9 (CLB, Central Laboratory of the Netherlands,
Red Cross Blood Transfusion Service) and 80H3 (Immunotech,
Marseille, France), respectively. The concentration of each of the
monoclonal antibodies against CD14, CD16, CD66e and CD66b contained
in the antibody composition is between about 0.5 to 5 .mu.g/ml,
preferably 2-3 .mu.g/ml.
[0072] Monoclonal antibodies against CD45RA, CD38 and CD36 are used
to label T-cells, B-cells plasma cells, granulocytes, platelets,
monocytes, differentiated erythroid precursors, and some committed
mature progenitors, to further enrich for stem cells. Examples of
monoclonal antibodies against CD45RA, CD38 and CD36 are 8D2.2
(StemCell Technologies, Vancouver, Canada, Craig et al., 1994,
British Journal of Haematology, 88:24-30.), Leu-18 (Becton
Dickinson, Mountain View, Calif.); T16 (Immunotech, Marseille,
France); and, FA60152 (Immunotech, Marseille, France) and IVC7
(CLB, Central Laboratory of the Netherlands Red Cross Blood
Transfusion Service), respectively. The concentration of each of
the monoclonal antibodies against CD45RA and CD36 contained in the
antibody composition is between about 0.5 to 5 .mu.g/ml, preferably
1 to 3 .mu.g/ml.
[0073] Table 2 sets out the most preferred monoclonal antibodies
specific for differentiated cells, their sources and
concentrations, for use in the antibody compositions of the
invention. Table 4 sets out the most preferred monoclonal
antibodies specific for tumor cells, and commercial
sources/references for the antibodies.
[0074] In one embodiment of the invention the antibody composition
for enriching for hematopoietic stem cells and progenitor cells,
comprises 2B7.1 (glycophorin A), SK7 (CD3), 32D12 (CD24), MEM54
(CD16), and MEM15 (CD14).
[0075] In another embodiment of the invention the antibody
composition for enriching for hematopoietic stem cells and
progenitor cells, comprises 2B7.1 (glycophorin A), SK7 (CD3), 32D12
(CD24), MEM54 (CD16), MEM15 (CD14), 6F10.2 (CD2), T199 (CD56),
J4.119 (CD19) and/or 80H3 (CD66b).
[0076] In further embodiment of the invention the antibody
composition for enriching for early hematopoietic stem and
progenitor cells comprises the monoclonal antibodies designated
2B7.1 (glycophorin A), SK7 (CD3), MEM15 (CD14), MEM154 (CD16),
32D12 (CD24), 80H3 (CD66b), J4.119 (CD19), 6F10.3 (CD2), MY31
(CD56), 8D2.2 (CD45RA), T16 (CD38) and FA60152 (CD36), or comprises
the monoclonal antibodies designated 10F7MN (glycophorin A), UCHT1
(CD3), ALB9 (CD24), 3G8 (CD16), MEM15 (CD14), B13.9 (CD66b), T199
(CD56), 6F10.3 (CD2), J4.119 (CD19), 8D2.2 (CD45RA), T16 (CD38) and
1VC7 (CD36).
[0077] A preferred antibody composition for removing differentiated
hematopoietic cells and breast and lung carcinoma cells from a
sample comprises the monoclonal antibodies 2B7.1 (glycophorin A),
SK7 (CD3), MEM15 (CD14), 3G8 (CD16), ALB9 (CD24), 80H3 (CD66b),
J4.119 (CD19), 6F10.3 (CD2), MY31 (CD56), or the monoclonal
antibodies 10F7MN (glycophorin A), SK7 (CD3), 32D12 (CD24), MEM154
(CD16), MEM15 (CD14), 80H3 (CD66b) or B13.9 (CD66b), T199 (CD56),
6F10.3 (CD2), J4.119 (CD19), and one or more of the monoclonal
antibodies specific for an antigen on the surface of a breast or
lung carcinoma as set forth in Table 4. Most preferably the
monoclonal antibodies specific for an antigen on the surface of
cells from a breast carcinoma used in a composition of the
invention are one or more of 5E11, H23A, 6E7, RAR, BerEp4 and
BRST1.
[0078] Preferred antibody compositions for enriching for
non-hematopoietic metastatic tumor cells from a sample containing
hematopoietic cells and non-hematopoietic metastatic tumor cells
comprise the monoclonal antibodies 2B7.1 (glycophorin A); MEM15
(CD14); 3G8 (CD16); 80H3 (CD66b); 6F10.3 (CD2); T16 (CD38); and
MEM28 (CD45).
[0079] Antibody compositions in accordance with the present
invention may be prepared which lack antibodies to a specific
differentiated cell type or lineage committed cell. For example, an
antibody composition may be prepared which does not contain
antibodies to the CD14, and CD16 antigens which are expressed on
monocytes. This composition may be used to prepare a cell
preparation which is enriched for monocytes. Other examples of
antibody compositions which can be used to prepare cell populations
enriched for monocytes, B-cells, T-cells, CD4.sup.+ T-cells,
CD8.sup.+ T-cells, and NK cells are set out in Table 3.
[0080] III. Process for Preparing Enriched Cell Preparations
[0081] The antibody compositions of the invention may be used to
enrich and recover cell preparations enriched in a specific cell
type such as stem cells and progenitor cells or non-hematopoietic
tumor cells. In accordance with a process of the invention, a
sample is reacted with an antibody composition containing
antibodies which are specific for selected antigens on the surface
of the cells to be removed from the sample and not on the cells to
be enriched in the sample, under suitable conditions, conjugates
form between the antibodies contained in the antibody composition
and the cells in the sample containing the antigens on their
surface; and the conjugates are removed to provide a cell
preparation enriched in specific cells.
[0082] (a) Progenitor Cell Enrichment
[0083] In one aspect the present invention provides a negative
selection process for enriching and recovering human hematopoietic
progenitor cells and stem cells in a sample containing human
hematopoietic differentiated, progenitor, and stem cells comprising
(a) reacting the sample with an antibody composition containing
antibodies capable of binding to the antigens glycophorin A, CD3
CD24, CD16, and CD14 under conditions so that conjugates are formed
between the antibodies and cells in the sample containing the
antigens glycophorin A, CD3 CD24, CD16, and CD14 on their surfaces;
(b) removing the conjugates; and, (c) recovering a cell preparation
which is enriched in human hematopoietic progenitor cells and stem
cells.
[0084] The antibody composition for enriching for progenitor and
stem cells may additionally include other antibodies specific for
antigens on differentiated cells such as CD2, CD56, CD19, CD66e
and/or CD66b. The selection of antibodies can largely depend on the
nature of the sample to be enriched. When the sample is fresh bone
marrow, the composition preferably comprises antibodies to
glycophorin A, CD3, CD24, CD16, CD14, CD66e and CD66b. When the
sample is mobilized peripheral blood or cord blood, the composition
preferably comprises glycophorin A, CD3, CD24, CD16, CD14, CD66e,
CD66b, CD56, CD2, and CD19. To enrich for early progenitor cells
the composition preferably includes glycophorin A, CD3, CD24, CD16,
CD14, CD66e, CD66b, CD56, CD2, CD19, CD45RA, CD36 and CD38.
[0085] The inventors have demonstrated that their method of
progenitor enrichment provides a cell preparation with greater than
50% recovery of progenitor and stem cells and 3 long depletion of
differentiated cells.
[0086] (b) Tumor Cell Depletion
[0087] In another aspect, the present invention provides a negative
selection process for enriching and recovering normal human
hematopoietic progenitor cells and stem cells and depleting
hematopoietic tumor cells in a sample containing human
hematopoietic differentiated, progenitor, and stem cells, and tumor
cells comprising (a) reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD3, CD24, CD16, and CD14, under conditions
so that conjugates are formed between the antibodies and the cells
in the sample having the antigens glycophorin A, CD3 CD24, CD16 and
CD14; (b) removing the conjugates; and (c) recovering a cell
preparation which is enriched in normal human hematopoietic
progenitor cells and stem cells and depleted in hematopoietic tumor
cells.
[0088] The present invention further provides a process for
enriching and recovering human hematopoietic stem cells and
progenitor cells and depleting tumor cells in a sample containing
differentiated cells, progenitor cells, stem cells and tumor cells,
comprising (a) reacting the sample with an antibody composition
containing antibodies capable of binding to the antigens
glycophorin A, CD3 CD24, CD16, and CD14, and an antigen present on
the tumor cells and optionally CD2, CD56, CD19, CD66e and/or CD66b
under conditions permitting the formation of conjugates between the
antibodies and cells in the sample having the antigens glycophorin
A, CD3 CD24, CD16, and CD14, and an antigen present on the tumor
cells and optionally CD2, CD56, CD19, CD66e and/or CD66b on their
surfaces; (b) removing the conjugates; and (c) recovering a cell
preparation which is enriched in human hematopoietic progenitor
cells and stem cells and depleted in tumor cells.
[0089] The inventors have demonstrated that the method of tumor
cell depletion provides a cell preparation with a 3 log depletion
of tumor cells.
[0090] (c) Tumor Cell Enrichment
[0091] In another aspect, the present invention provides a negative
selection process for enriching for non-hematopoietic metastatic
tumor cells in a sample containing the tumor cells and
hematopoietic cells comprising (a) reacting the sample with an
antibody composition comprising antibodies specific for glycophorin
A, CD2, CD14, CD16, CD38, CD45 and CD66b under conditions so that
conjugates are formed between the antibodies and hematopoietic
cells in the sample expressing the antigens glycophorin A, CD2,
CD14, CD16, CD38, CD45 and CD66b; (b) removing the conjugates; and
(c) recovering a cell preparation enriched in the tumor cells.
[0092] In one embodiment, the tumor cells are metastatic tumor
cells derived from epithelial cancers of the bronchi, mammary
ducts, reproductive system, gastrointestinal tract and urogenital
tract such as lung carcinoma, breast carcinoma, colon carcinoma,
prostate carcinoma and bladder carcinoma.
[0093] The inventors have demonstrated that their method of tumor
cell enrichment provides a cell preparation that is enriched at
least 2 log, generally 3-4 log, in tumor cells. The tumor enriched
cell preparations can be used to detect metastatic tumor cells in
sample. Accordingly, the present invention also provides a method
of detecting tumor metastasis in a sample comprising (a) reacting
the sample with an antibody composition comprising antibodies
specific for glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b
under conditions so that conjugates are formed between the
antibodies and hematopoietic cells in the sample expressing the
antigens glycophorin A, CD2, CD14, CD16, CD38, CD45 and CD66b; (b)
removing the conjugates; and (c) recovering a cell preparation
enriched in the tumor cells; and (d) detecting the tumor cells in
the cell preparation. The tumor cells may be detected using
techniques known in the art. For example, antibodies specific for
tumor cells may be used in antibody mediated detection methods such
as immuno-cytochemical staining (ICC).
[0094] In all of the above negative selection processes for cell
enrichment, conditions which permit the formation of conjugates may
be selected having regard to factors such as the nature and amounts
of the antibodies in the antibody composition, and the estimated
concentration of targeted cells in the sample.
[0095] The antibodies in the antibody compositions may be labelled
with a marker or they may be conjugated to a matrix. Examples of
markers are biotin, which can be removed by avidin bound to a
support, and fluorochromes, e.g. fluorescein, which provide for
separation using fluorescence activated sorters. Examples of
matrices are magnetic beads, which allow for direct magnetic
separation (Kernshead 1992), panning surfaces e.g. plates,
(Lebkowski, J. S, et al., (1994), J. of Cellular Biochemistry
supple. 18b:58), dense particles for density centrifugation (Van
Vlasselaer, P., Density Adjusted Cell Sorting (DACS), A Novel
Method to Remove Tumor Cells From Peripheral Blood and Bone Marrow
StemCell Transplants. (1995) 3rd International Symposium on Recent
Advances in Hematopoietic Stem Cell Transplantation-Clinical
Progress, New Technologies and Gene Therapy, San Diego, Calif.),
dense particles alone (Zwerner et al., Immunol. Meth. 1996
198(2):199-202) adsorption columns (Berenson et al. 1986, Journal
of Immunological Methods 91:11-19.), and adsorption membranes. The
antibodies may also be joined to a cytotoxic agent such as
complement or a cytotoxin, to lyse or kill the targeted
differentiated or tumors cells.
[0096] The antibodies in the antibody compositions may be directly
or indirectly coupled to a matrix. For example, the antibodies in
the compositions of the invention may be chemically bound to the
surface of magnetic particles for example, using cyanogen bromide.
When the magnetic particles are reacted with a sample, conjugates
will form between the magnetic particles with bound antibodies
specific for antigens on the surfaces of the differentiated cells
and/or tumor cells, and the differentiated cells and/or tumor cells
having the antigens on their surfaces.
[0097] Alternatively, the antibodies may be indirectly conjugated
to a matrix using antibodies. For example, a matrix may be coated
with a second antibody having specificity for the antibodies in the
antibody composition. By way of example, if the antibodies in the
antibody composition are mouse IgG antibodies, the second antibody
may be rabbit anti-mouse IgG.
[0098] The antibodies in the antibody compositions may also be
incorporated in antibody reagents which indirectly conjugate to a
matrix. Examples of antibody reagents are bispecific antibodies,
tetrameric antibody complexes, and biotinylated antibodies.
[0099] Bispecific antibodies contain a variable region of an
antibody in an antibody composition of the invention, and a
variable region specific for at least one antigen on the surface of
a matrix. 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.
[0100] A tetrameric immunological complex may be prepared by mixing
a first monoclonal antibody which is capable of binding to at least
one antigen on the surface of a matrix, and a second monoclonal
antibody from the antibody composition of the invention. The first
and second monoclonal antibody are from a first animal species. The
first and second antibody are reacted with an about 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 equimolar amount of the F(ab').sub.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. Pat. No. 4,868,109 to Lansdorp,
which is incorporated herein by reference for a description of
tetrameric antibody complexes and methods for preparing same).
[0101] The antibodies of the invention may be biotinylated and
indirectly conjugated to a matrix which is labelled with (strept)
avidin. For example, biotinylated antibodies contained in the
antibody composition of the invention may be used in combination
with magnetic iron-dextran particles that are covalently labelled
with (strept) avidin (Miltenyi, S. et al., Cytometry 11:231, 1990).
Many alternative indirect ways to specifically cross-link the
antibodies in the antibody composition and matrices would also be
apparent to those skilled in the art.
[0102] In an embodiment of the invention, the cell conjugates are
removed by magnetic separation using magnetic particles. Suitable
magnetic particles include particles in ferrofluids and other
colloidal magnetic solutions. "Ferrofluid" refers to a colloidal
solution containing particles consisting of a magnetic core, such
as magnetite (Fe.sub.3O.sub.4) coated or embedded in material that
prevents the crystals from interacting. Examples of such materials
include proteins, such as ferritin, polysaccharides, such as
dextrans, or synthetic polymers such as sulfonated polystyrene
cross-linked with divinylbenzene. The core portion is generally too
small to hold a permanent magnetic field. The ferrofluids become
magnetized when placed in a magnetic field. Examples of ferrofluids
and methods for preparing them are described by Kernshead J. T.
(1992) in J. Hematotherapy, 1:35-44, at pages 36 to 39, and Ziolo
et al. Science (1994) 257:219 which are incorporated herein by
reference. Colloidal particles of dextran-iron complex are
preferably used in the process of the invention. (See Molday, R. S.
and McKenzie, L. L. FEBS Lett. 170:232, 1984; Miltenyi et al.,
Cytometry 11:231, 1990; and Molday, R. S. and MacKenzie, D., J.
Immunol. Methods 52:353, 1982; Thomas et al., J. Hematother. 2:297
(1993); and U.S. Pat. No. 4,452,733, which are each incorporated
herein by reference).
[0103] FIG. 1 is a schematic representation of magnetic cell
labeling using tetrameric antibody complexes and colloidal dextran
iron.
[0104] In accordance with the magnetic separation method, the
sample containing the progenitor and stem cells to be recovered, is
reacted with the above described antibody reagents, preferably
tetrameric antibody complexes, so that the antibody reagents bind
to the targeted differentiated cells and/or tumor cells present in
the sample to form cell conjugates of the targeted differentiated
cells and/or tumor cells and the antibody reagents. The reaction
conditions are selected to provide the desired level of binding of
the targeted differentiated cells and/or tumor cells and the
antibody reagents. Preferably the sample is incubated with the
antibody reagents for a period of 5 to 60 minutes at either
4.degree. or ambient room temperature. The concentration of the
antibody reagents is selected depending on the estimated
concentration of the targeted differentiated cells in the sample.
Generally, the concentration is between about 0.1 to 50 .mu.g/ml of
sample. The magnetic particles are then added and the mixture is
incubated for a period of about 5 minutes to 30 minutes at the
selected temperature. The sample is then ready to be separated over
a magnetic filter device. Preferably, the magnetic separation
procedure is carried out using the magnetic filter and methods
described in U.S. Pat. No. 5,514,340 to Lansdorp and Thomas which
is incorporated in its entirety herein by reference.
[0105] The sample containing the magnetically labelled cell
conjugates is passed through the magnetic filter in the presence of
a magnetic field. In a preferred embodiment of the invention, the
magnet is a dipole magnet with a gap varying from 0.3 to 3.0 inches
bore and having a magnetic field of 0.5-2 Tesla. The magnetically
labelled cell conjugates are retained in the high gradient magnetic
column and the materials which are not magnetically labelled flow
through the column after washing with a buffer.
[0106] The preparation containing non-magnetically labelled cells
may be analyzed using procedures such as flow cytometry. The
ability of the cells in the preparation to produce colony-forming
cells or long term culture initiating cells (LTCIC) in culture or
repopulate SCID mice in a SCID repopulating assay (SRC) may also be
assessed. The efficiency of the separation procedure may also be
determined by monitoring the recovery of CD34.sup.+ cells,
CD34.sup.+ CD38.sup.- cells and colony forming cells.
[0107] The antibody compositions of the invention may also be used
to prepare a cell preparation which is enriched for a specific
differentiated cell type. This is achieved by using antibody
compositions which lack antibodies to the specific differentiated
cell type, in the above described processes of the invention.
Particular embodiments of these processes of the invention are set
out below. It will be appreciated that the markers, matrices,
antibody reagents, and procedures described herein may be used in
these processes to facilitate recovery of cell preparations
enriched for a specific differentiated cell type. Examples of
antibodies which may be used in these processes are set out in
Table 3.
[0108] In accordance with one embodiment of the invention, a
process is provided for enriching and recovering monocytes from a
blood sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD2, CD3, CD56, and CD24 or CD19, and
optionally CD66b, under conditions so that cell conjugates are
formed between the antibodies and the cells in the sample having
the antigens glycophorin A, CD2, CD3, CD56, and CD24 or CD19 and
optionally CD66b on their surfaces; removing the cell conjugates;
and recovering a cell preparation which is enriched in
monocytes.
[0109] In accordance with another embodiment of the invention, a
process is provided for enriching and recovering monocytes from a
bone marrow sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD2, CD3, CD56, and CD24 or CD19, and
optionally CD66b and/or CD66e, under conditions so that cell
conjugates are formed between the antibodies and the cells in the
sample having the antigens glycophorin A, CD2, CD3, CD56, and CD24
or CD19 and optionally CD66b and/or CD66e on their surfaces;
removing the cell conjugates; and recovering a cell preparation
which is enriched in monocytes.
[0110] In accordance with another embodiment of the invention, a
process is provided for enriching and recovering B-cells from a
blood sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD2, CD3, CD56, CD16 and CD14, and
optionally CD66b under conditions so that cell conjugates are
formed between the antibodies and the cells in the sample having
the antigens glycophorin A, CD2, CD3, CD56, CD16 and CD14 and
optionally CD66b on their surfaces; removing the cell conjugates;
and recovering a cell preparation which is enriched in B-cells.
[0111] In accordance with another embodiment of the invention, a
process is provided for enriching and recovering B-cells from a
bone marrow sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD2, CD3, CD56, CD16 and CD14, and
optionally CD66b and/or CD66e under conditions so that cell
conjugates are formed between the antibodies and the cells in the
sample having the antigens glycophorin A, CD2, CD3, CD56, CD16 and
CD14 and optionally CD66b and/or CD66e on their surfaces; removing
the cell conjugates; and recovering a cell preparation which is
enriched in B-cells.
[0112] In accordance with another embodiment of the invention, a
process is provided for enriching and recovering T-cells from a
blood sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD16, CD14, CD19, CD56, and optionally
CD66b under conditions so that cell conjugates are formed between
the antibodies and the cells in the sample having the antigens
glycophorin A, CD16, CD14, CD19, CD56, and optionally CD66b on
their surfaces; removing the cell conjugates; and recovering a cell
preparation which is enriched in T-cells.
[0113] In accordance with another embodiment of the invention, a
process is provided for enriching and recovering T-cells from a
bone marrow sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD16, CD14, CD19, CD56, and optionally
CD66b and/or CD66e under conditions so that cell conjugates are
formed between the antibodies and the cells in the sample having
the antigens glycophorin A, CD16, CD14, CD19, CD56, and optionally
CD66b and/or CD66e on their surfaces; removing the cell conjugates;
and recovering a cell preparation which is enriched in T-cells.
[0114] In accordance with yet another embodiment of the invention,
a process is provided for enriching and recovering CD4.sup.+T-cells
from a blood sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD16, CD14 CD19, CD56, CD8, and optionally
CD66b, under conditions so that cell conjugates are formed between
the antibodies and the cells in the sample having the antigens
glycophorin A, CD16, CD14, CD19, CD56, CD8, and optionally CD66b on
their surfaces; removing the cell conjugates; and recovering a cell
preparation which is enriched in CD4.sup.+T-cells.
[0115] In accordance with yet another embodiment of the invention,
a process is provided for enriching and recovering CD4.sup.+T-cells
from a bone marrow sample comprising reacting the sample with an
antibody composition containing antibodies capable of binding to
the antigens glycophorin A, CD16, CD14 CD19, CD56, CD8, and
optionally CD66b and/or CD66e, under conditions so that cell
conjugates are formed between the antibodies and the cells in the
sample having the antigens glycophorin A, CD16, CD14, CD19, CD56,
CD8, and optionally CD66b and/or CD66e on their surfaces; removing
the cell conjugates; and recovering a cell preparation which is
enriched in CD4.sup.+T-cells.
[0116] In accordance with a further embodiment of the invention, a
process is provided for enriching and recovering CD8.sup.+T-cells
from a blood sample comprising reacting the sample with an antibody
composition containing antibodies capable of binding to the
antigens glycophorin A, CD16, CD14, CD19, CD56, CD4, and optionally
CD66b under conditions so that cell conjugates are formed between
the antibodies and the cells in the sample having the antigens
glycophorin A, CD16, CD14, CD19, CD56, CD4, and optionally CD66b on
their surfaces; removing the cell conjugates; and recovering a cell
preparation which is enriched in CD8.sup.+T-cells.
[0117] In accordance with a further embodiment of the invention, a
process is provided for enriching and recovering CD8.sup.+T-cells
from a bone marrow sample comprising reacting the sample with an
antibody composition containing antibodies capable of binding to
the antigens glycophorin A, CD16, CD14, CD19, CD56, CD4, and
optionally CD66b and/or CD66e under conditions so that cell
conjugates are formed between the antibodies and the cells in the
sample having the antigens glycophorin A, CD16, CD14, CD19, CD56,
CD4, and optionally CD66b and/or CD66e on their surfaces; removing
the cell conjugates; and recovering a cell preparation which is
enriched in CD8.sup.+T-cells.
[0118] In accordance with a still further embodiment of the
invention, a process is provided for enriching and recovering
NK-cells from a blood sample comprising reacting the sample with an
antibody composition containing antibodies capable of binding to
the antigens glycophorin A, CD4, CD14, CD19, and CD3, and
optionally CD66b under conditions so that cell conjugates are
formed between the antibodies and the cells in the sample having
the antigens glycophorin A, CD4 CD14, CD19, and CD3, and optionally
CD66b on their surfaces; removing the cell conjugates; and
recovering a cell preparation which is enriched in NK-cells.
[0119] In accordance with a still further embodiment of the
invention, a process is provided for enriching and recovering
NK-cells from a bone marrow sample comprising reacting the sample
with an antibody composition containing antibodies capable of
binding to the antigens glycophorin A, CD4 CD14, CD19, and CD3, and
optionally CD66b and/or CD66e under conditions so that cell
conjugates are formed between the antibodies and the cells in the
sample having the antigens glycophorin A, CD4, CD14, CD19, and CD3,
and optionally CD66b and/or CD66e on their surfaces; removing the
cell conjugates; and recovering a cell preparation which is
enriched in NK-cells.
[0120] In accordance with another embodiment, a process is provided
for enriching and recovering basophils from whole blood sample
comprising reacting the sample with an antibody composition
containing antibodies capable of binding to the antigens
glycophorin A, CD2, CD3, CD14, CD15, CD16, CD19, CD24, CD34, CD36,
CD56 and CD45RA under conditions so that cell conjugates are formed
between the antibodies and the cells in the sample having the
antigens glycophorin A, CD2, CD3, CD14, CD15, CD16, CD19, CD24,
CD34, CD36, CD56 and CD45RA on their surfaces; removing the cell
conjugates; and recovering a cell preparation which is enriched in
basophils.
[0121] In accordance with a further embodiment, a process is
provided for enriching and recovering dendritic cells from a blood
sample comprising reacting the sample with an antibody composition
containing antibodies capable of binding to the antigens
glycophorin A, CD3, CD14, CD16, CD19, CD34, CD56 and CD66b under
conditions so that cell conjugates are formed between the
antibodies and the cells in the sample having the antigens
glycophorin A, CD3, CD14, CD16, CD19, CD34, CD56 and CD66b on their
surfaces; removing the cell conjugates; and recovering a cell
preparation which is enriched in dendritic cells. Dentritic can
also be generated by culturing cells enriched for progenitors or
monocytes using the previously mentioned enrichment cocktails.
[0122] In accordance with yet a further embodiment, a process is
provided for enriching and recovering granulocytes from whole blood
sample comprising reacting the sample with an antibody composition
containing antibodies capable of binding to the antigens
glycophorin A, CD2, CD56, CD19, CD14 and CD3 under conditions so
that cell conjugates are formed between the antibodies and the
cells in the sample having the antigens glycophorin A, CD2, CD56,
CD19, CD14 and CD3 on their surfaces; removing the cell conjugates;
and recovering a cell preparation which is enriched in
granulocytes.
[0123] IV. Uses of the Compositions and Processes of the
Invention
[0124] The compositions and processes of the invention may be used
in the processing of biological samples including blood in
particular, cord blood and whole blood. It has also been found that
the antibody compositions of the invention can be used to prepare
hematopoietic progenitor and stem cell preparations from bone
marrow samples, including previously frozen bone marrow
samples.
[0125] The processes of the invention are preferably used to
deplete or purge erythrocytes, 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 processes described herein.
[0126] Using the process of the invention it is possible to recover
a highly purified preparation of human hematopoietic
stem/progenitor cells. In particular, a hematopoietic cell
population containing greater than 50% of the hematopoietic
progenitor/stem cells present in the original sample, and which is
depleted of differentiated cells and/or tumor cells in the original
sample by greater than 3 logarithms may be obtained. 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.
[0127] The processes and compositions of the invention permit the
isolation and recovery of mature dendritic cells and their
precursors from blood (Horrocks et al., In press.). 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.
[0128] The cell preparations obtained using the processes of the
invention may be used to isolate and evaluate factors associated
with the differentiation and maturation of human hematopoietic
cells. The cell preparations may also be used to determine the
effect of a substance on cell growth and/or differentiation into a
particular lineage
[0129] The antibody compositions and processes 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. 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 and T-cells may also be used in immune therapy
against certain malignancies.
[0130] The tumor-enriching antibody composition of the invention is
adapted to enrich for tumor cells, in particular non-hematopoietic
metastatic tumor cells. The composition 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. The tumor enriching antibody composition applied in one
step to a sample of peripheral blood, frozen peripheral blood, or
bone marrow containing tumor cells results at least a 2 log
enrichment (and typically 3-4 log) of the tumor cells.
[0131] One currently used method for enriching for
non-hematopoietic tumor cells is to use a negative selection
technique with antibodies specific for CD45. The inventors have
compared their antibody composition with anti-CD45 alone on the
ability to enrich peripheral blood mononuclear cells for breast
carcinoma tumor cells and have shown that the antibody composition
of the invention enriches the tumor cells 10 fold (1 log) over
anti-CD45 alone.
[0132] The present invention also includes a useful kit in
preparing a cell preparation enriched in hematopoietic stem cells
and progenitor cells comprising antibodies specific for glycophorin
A, CD3, CD24, CD16, CD14 and instructions for preparing a cell
preparation enriched in hematopoietic stem cells and progenitor
cells.
[0133] The present invention further includes a kit useful in
preparing a cell preparation enriched in hemotopoietic stem and
progenitor cells and depleted in hematopoietic tumor cells
comprising antibodies specific for glycophorin A, CD3, CD24, CD16,
CD14 and instructions for preparing a cell preparation enriched in
hematopoietic stem cells and progenitor cells and depleted in
hematopoietic tumor cells.
[0134] The present invention also includes a kit useful in
preparing a cell preparation enriched in hemotopoietic stem cells
and progenitor cells and depleted in tumor cells comprising
antibodies specific for glycophorin A, CD3, CD24, CD16, CD14, and
an antigen present on the tumor cells and instructions for
preparing a cell preparation enriched in hematopoietic stem cells
and progenitor cells and depleted in tumor cells.
[0135] The present invention also relates to kits useful in
preparing a preparation of non-hematopoietic tumor cells comprising
antibodies specific for glycophorin A, CD2, CD14, CD16, CD38, CD45
and CD66b and instructions for performing the tumor cell enriching
processes of the invention.
[0136] The following non-limiting examples are illustrative of the
present invention:
EXAMPLES
Example 1
[0137] Method for Evaluating Antibody Combinations
[0138] Suspensions of normal human bone marrow, human cord blood,
mobilized human peripheral blood and previously frozen human bone
marrow were labelled with tetrameric antibodies and colloidal
dextran iron for magnetic cell depletions. Monoclonal antibodies
recognizing lineage specific cell surface antigens were mixed with
a mouse IgG.sub.1 anti-dextran antibody (Thomas, T. E, et al.
(1992), J. Immunol Methods 154:245;252) and a rat IgG.sub.1
monoclonal antibody which recognizes the Fc portion of the mouse
IgG.sub.1 molecule (TFL-P9) (Lansdorp, P. M, and Thomas, T. E.
(1990), Mol. Immunol. 27:659-666). Tetrameric antibody complexes
(Lansdorp, P. M, and Thomas, T. E. (1990), Mol. Immunol.
27:659-666; U.S. Pat. No. 4,868,109 to Lansdorp) spontaneously form
when mouse IgG.sub.1 molecules (the lineage specific monoclonal
antibody and anti-dextran) are mixed with P9. A proportion of these
tetrameric antibody complexes are bifunctional, recognizing an
antigen on the surface of the target cell on one side and dextran
(part of the magnetic colloidal dextran iron) on the other.
Tetrameric antibody complexes were made for all the antibodies in
the lineage cocktail. FIG. 1 shows a schematic representation of
magnetic cell labeling using tetrameric antibody complexes and
colloidal dextran iron.
[0139] Cells were labelled for separation (1-5.times.10.sup.7
cells/ml) by incubating them with the desired combination of
tetramers for 30 min on ice followed by a 30 min incubation with
colloidal dextran iron (final OD450=0.6) (Molday and MacKenzie
1982, 52(3): 353-367). The cells were then passed through a
magnetic filter (U.S. Pat. No. 5,514,340; inventors Lansdorp and
Thomas) at 1 cm/min. The magnetically labelled cells bind to the
filter and the unlabeled cells pass through. FIG. 1 shows a
schematic representation of magnetic cell labeling using tetrameric
antibody complexes and colloidal dextran iron.
[0140] The flow through fraction is collected and analyzed for
hematopoietic colony forming cells (CFU-GM, CFU-C, LTCIC) (Eaves,
C. J. and Eaves, A. J. 1992 In: Current Therapy in
Hematology-Oncology, Fourth Edition pp. 159-167), CD34+ cells, and
CD34+ CD38- cells. The enrichment of these cell types depends on
how well the antibody cocktail has targeted other cells for
removal. Each antibody cocktail was evaluated for the purity and
recovery of colony forming cells, CD34+ cells, and CD34+CD38-
cells. FIG. 2 shows a FACS histogram of mobilized peripheral blood
before and after progenitor enrichment via lineage depletion.
Example 2
[0141] Antibodies for the Enrichment of Progenitor Cells
(Progenitor Cocktail)
[0142] The results of numerous cell separations identified a
combination of lineage specific antibodies that produce the maximum
enrichment and recovery of CD34+ cells and colony forming
cells.
[0143] Targeting Erythrocytes--Anti-glycophorin A antibodies were
used to label erythrocytes for depletion. Many of these antibodies
will cause agglutination at moderate to high antibody
concentrations (3-10 .mu.g/ml). It was found that cells could be
effectively targeted for magnetic depletion with concentrations of
anti-glycophorin antibody that were several fold lower than that
which caused agglutination.
[0144] Targeting Lymphocytes--B, T, and NK cells were targeted with
monoclonal antibodies against CD24, CD3, CD19, CD20, CD56, CD2.
Initial depletions of mobilized peripheral blood using just
anti-CD24 and CD3 for lymphocyte depletion showed that a proportion
of the CD34 negative cells in the purified fraction were CD56
positive (NK cells). Subsequent tests with and without anti-CD56
increased the purity of CD34+ cells in the recovered fraction by
12-20%. Adding an anti-CD2 to the cocktail increased the purity an
additional 12-13%. Anti-CD2 and anti-CD56 had no significant effect
on lineage depletions of fresh bone marrow. It is likely that bone
marrow does not have as many CD3- CD2+ and CD3-CD56+ cells.
Anti-CD2 and anti-CD56 are added to the depletion cocktails for
mobilized peripheral blood but not bone marrow.
[0145] Antibodies against CD24 were sufficient to target all
detectable B-cells for depletion. Adding anti-CD19 gave no
additional enrichment of CD34+ cells from mobilized peripheral
blood or bone marrow. Substituting CD19 for CD24 in separations of
fresh bone marrow had no effect on the enrichment or recovery of
CD34+ cells, hematopoietic colony forming cells and LTCIC.
Replacing anti-CD24 with anti-CD20 or both anti-CD19 and anti-CD20
had no significant effect on separations of mobilized peripheral
blood. An effect was seen in a separation with cord blood; when
anti-CD24 was replaced with anti-CD19, the purity was decreased
21%, but recovery of CD34+ cells was increased 28%.
[0146] Targeting Mature Myeloid Cells--Monocytes were effectively
targeted with an antibody against CD14 in all cell suspensions
tested. The removal of granulocytes from peripheral blood and fresh
bone marrow was more efficient using both anti-CD16 and anti-CD66b
rather than anti-CD16 alone and adding anti-CD66e gave an
additional 10% enrichment of CD34+ cells from fresh bone marrow and
20% enrichment for peripheral blood. Anti-CD16 alone was sufficient
to deplete the granulocytes from previously frozen marrow. Adding
anti-CD41 or CD42a did not increase the purity of CD34+ cells in
either peripheral blood or bone marrow.
Example 3
[0147] Antibodies for the Enrichment of Stem Cells (Stem Cell
Cocktail)
[0148] Both pluripotent stem cells and committed progenitors
express CD34, but the CD34 compartment can be further subdivided
using a variety of cell surface markers to isolate these cell
types. Stem cells co-purify in a population of CD34.sup.+ cells
which lack or have low expression of certain lineage markers (CD38,
CD33, CD45RA, CD71, CD36 and HLA-DR) (Craig et al. 1994, British
Journal of Haematology, 88:24-30; Lansdorp, P. A I. and Dragowska,
W. 1992, J. Exp. Med. 175:1501-1509; Sutherland, H, J., et al.
1989, Blood 74.1563-1570.). If antibodies recognizing these
antigens are included in the lineage cocktail one can further
enrich for stem cells while losing some of the committed mature
CD34.sup.+ cells. Antibodies to CD36, CD38 and CD45RA were added to
the lineage cocktail to specifically enrich for stem cells. The
recovery of CD34+ CD38.sup.- cells (Tables 5) and LTCICs (Table 6)
were monitored to determine the efficiency of the lineage
depletions with the "stem cell cocktail".
[0149] Including anti-CD45RA in the stem cell cocktail does not
negate the need for anti-CD24 in the cocktail nor does anti-CD36
allow the removal of CD66e. Adding anti-CD36 did increase the
purity of CD34.sup.+CD38.sup.- cells in separations of previously
frozen bone marrow by 15%. The addition of anti-transferrin (CD71)
antibody to the cocktail resulted in very poor recovery of CD34+
CD38- cells, and LTCIC as well as producing a significant number of
dead cells in the enriched fraction (viability normally
>95%).
[0150] The results shown in Tables 5 and 6 demonstrate that
separation of blood and bone marrow with the stem cell cocktail
produced a cell suspension which is at least 30% and up to about
80% CD34+ CD38- with up to 90% recovery of these cells. FIG. 3
shows a FACS profile of peripheral blood before and after
progenitor enrichment with the antibody cocktail.
Example 4
[0151] Different Antibodies to the Same Antigen
[0152] The enrichment of hematopoietic progenitor cells via lineage
depletion is not only dependent on the number of types of committed
cells that are targeted but also the effectiveness of this
targeting and subsequent removal using magnetic separation or other
antibody mediated techniques. It was found that different
antibodies recognizing the same antigen may reproducibly produce
different degrees of progenitor enrichment. The anti-CD24 antibody
32D12 produced better results in lineage depletions than ALB9 (also
anti-CD24); the purity of the enriched cell suspension increased
10% in a separation with cord blood. In cell depletions with a
single tetramer type, 32D12 out performed ALB9 and anti-glycophorin
antibody 10F7MN out performed anti-glycophorin antibody D2.10
although switching anti-glycophorin antibodies in a lineage
depletion had no significant effect.
[0153] The criteria for choosing a particular antibody at a given
concentration is its performance in a magnetic cell separation
which equates to the maximum depletion of antibody targeted cells
with the maximum recovery of non-target cells. Often depletion of
anybody targeted cells increases with antibody concentration but so
does the non-specific labeling of cells. In general, the result
looked for was a 3 log depletion with greater than 75% recovery of
CD34+ cells or non-targeted lymphocytes if the test cell suspension
was steady state peripheral blood. The performance in a cell
separation typically mimicked the degree of specific cell labeling
and the degree on non-specific labeling measured by FACS (sheep
anti-mouse FITC staining). Staining experiments were often run to
eliminate antibodies (specific staining low, non-specific staining
high) and reduce the number of antibody concentrations to be tested
in cell separations.
Example 5
[0154] Purging Breast Carcinoma Cells (BT20 or T47D Cells)
[0155] Tetramers of anti-breast carcinoma antibodies as shown in
Table 4 were combined with a progenitor enrichment cocktail (D2.10,
UCHT1, MEM15, 3G8, ALB9, 80H3, J4.119, 6F10.3, T199, and optionally
8D2.2, T16 and FA60152, or 10F7MN, UCHT1, 32D12, MEM154, MEM15 or
B13.9, T199, 6F10.3, J4.119, and optionally, 8D2.2, T16 and 1VC7)
to produce a cocktail for breast carcinoma purging and debulking.
Including the lineage depletion increases the degree of tumor purge
over that seen with just anti-tumor antibodies alone (Table 7).
Breast carcinoma cell lines were added to previously frozen marrow,
peripheral blood leukapheresis or fresh bone marrow. Tumor cell
purges were performed using the anti-breast carcinoma antibodies
indicated in Table 7 with and without the standard lineage
depletion (progenitor enrichment cocktail). The recovery of
hematopoietic progenitors during lineage depletion is given in
Table 8. Enrichment of progenitors was generally 50 to 100
fold.
[0156] In summary, purging tumor cells for hematopoietic
progenitors in a one step selection using the antibody cocktail as
indicated in Table 7 achieves a much higher degree of tumor cell
purging than positive selection techniques while offering a similar
degree of progenitor enrichment. The recoveries of hematopoietic
progenitor cells in a lineage depletion are greater than those
typically seen with positive selection.
Example 6
[0157] Enrichment of Breast Carcinoma Cells in Bone Marrow
[0158] Cells from the CAMA breast carcinoma cell line were mixed
with previously frozen bone marrow (BM) and processed with the
enrichment antibody composition (D2.10, UCHT1, MEM15, 3G8, 8OH3,
J4119, 6F10.3, T199, 8D2.2, T16, FA6.152, and J33) in a one step
magnetic depletion. The results shown in Table 9 demonstrates that
the CAMA cells were enriched 2-3 log using the tumor enrichment
antibody compositions.
Example 7
[0159] Enrichment of Breast Carcinoma Cells in Peripheral Blood
[0160] Cells from the CAMA breast carcinoma cell line were seeded
into previously frozen peripheral blood mononuclear cells (PBMC)
and processed with the enrichment antibodies capable of binding to
glycophorin A (2B7.1), CD2 (6710.3), CD14 (MEM15), CD16 (3G8), CD38
(T16), CD45 (J33) and CD66b (80H3) in a one step magnetic
depletion. The results shown in Table 10 demonstrate that CAMA
cells were enriched up to 4.5 log.
Example 8
[0161] Comparison with Antibodies to CD45
[0162] Cells from the CAMA breast carcinoma cell line were seeded
into previously frozen peripheral blood mononuclear cells (PBMC)
and processed with the enrichment antibodies capable of binding to
glycophorin A (2B7.1), CD2 (6710.3), CD14 (MEM15), CD16 (3G8), CD38
(T16), CD45 (J33) and CD66b (80H3). The results were compared with
the common method of negative selection, ie. the use of anti-CD-45
alone. The results shown in Table 11, demonstrates that there is
close to a ten fold (1 log) greater enrichment using the antibody
composition of the invention, over negative selection with
CD45.
Example 9
[0163] Enrichment of Epithelial Tumor Cells from Pleural Effusion
Samples
[0164] Pleural effusion samples were taken from patients with
suspected metastatic disease. The pleural effusions were separated
using the tumor enrichment composition of antibodies capable of
binding to glycophorin A (2B7.1), CD2 (6710.3), CD14 (MEM15), CD16
(3G8), CD38 (T16), CD45 (J33) and CD66b (80H3). As shown in Table
12, there is up to a 2.5 log enrichment using the antibody
composition of the invention.
Example 10
[0165] Enrichment of Epithelial Tumors Cells from Pleural Effusion
Samples Diluted into Peripheral Blood
[0166] Pleural effusion samples were taken from patients with
suspected metastatic disease and seeded into previously frozen PBMC
to mimic metastatic cells in the blood, and then separated using
the tumor enrichment composition of antibodies capable of binding
to glycophorin A (2B7.1), CD2 (6710.3), CD14 (MEM15), CD16 (3G8),
CD38 (T16), CD45 (J33) and CD66b (80H3). As shown in Table 13,
there is up to a enrichment using the antibody composition of the
invention, with up to a 4.9 log enrichment and 95% recovery.
[0167] While what is shown and described herein constitutes various
preferred embodiments of the subject invention, it will be
understood that various changes can be made to such embodiments
without departing from the subject invention, the scope of which is
defined in the appended claims.
1TABLE 1 Optimal Antibody Cocktail for the Enrichment of
Hematopoietic Progenitors Cell optimum antibody % purity % recovery
Suspension cocktail anti- CD34+ cells CD34+ cells fresh bone gly*,
CD3, CD24, 39, 44, 38 67, 48, 55 marrow CD16, CD14, CD66e, CD66b,
previously gly, CD3, CD24, 64, 46, 50, 53 85, 55, 82, 64 frozen
bone CD16, CD14, marrow mobilized gly, CD3, CD24, 51, 50, 57 43,
49, 85 peripheral CD16, CD14, blood CD66e, CD66b, CD56, CD2, CD19
cord blood gly, CD3, CD24, 56, 88, 58, 75, 85, 53, CD16, CD14, 55,
63 67, 48 CD66e, CD66b, CD56, CD2, CD19 *gly = glycophorin A
[0168]
2TABLE 2 Antibodies used in Lineage Depletions Concentration
Antigen Antibody Source ug/ml glycophorin 10F7MN* U.S. Pat. No.
4,752,582 1 A D2.10 IMMUNOTECH, Marseille, France 2 2B7.1 StemCell
Technologies 1 CD2 6F10.3 IMMUNOTECH, Marseille, France 3 CD3 UCHT1
IMMUNOTECH, Marseille, France 3 SK7 Becton Dickinson
Immunocytometry, Mountain View, Calif. CD4 13B8.2 Becton Dickinson
Immunocytometry, Mountain View, Calif. 3 CD8 B911 Becton Dickinson
Immunocytometry, Mountain View, Calif. 3 OKT3 BioDesigns 3 CD14 MEM
15 Dr. Vaclav Horejsi, Institute of Molecular 2 MEM 18 Genetics
Academy of Sciences of the Czech Republic, Praha, Czech 2 Republic;
Cedarlane Laboratories Hornby, Ontario, Canada CD16 MEM 154* Dr.
Vaclav Horejsi, Institute of Molecular 2 Genetics Academy of
Sciences of the Czech Republic, Praha, Czech Republic; Cedarlane
Laboratories Hornby, Ontario, Canada 3G8 IMMUNOTECH, Marseille,
France 3 NKP15 Becton Dickinson Immunocytometry, Mountain View,
Calif. 3 CD19 J4.119 IMMUNOTECH, Marseille, France 3 4G7 Becton
Dickinson Immunocytometry, Mountain View, Calif. CD20 MEM97 Dr.
Vaclav Horejsi, Institute of Molecular 3 Genetics Academy of
Sciences of the Czech Republic, Praha, Czech Republic; Cedarlane
Laboratories Hornby, Ontario, Canada L27 Becton Dickinson
Immunocytometry, Mountain View, Calif. 3 CD24 32D12* Dr. Steinar
Funderud, Institute for Cancer 2 Research, Dept. of Immunology,
Oslo, Norway ALB9 IMMUNOTECH, Marseille, France 3 CD36 FA60152
IMMUNOTECH, Marseille, France 3 IVC7 CLB, Central Laboratory of the
Netherlands, Red Cross Blood Transfusion Service CD38 T16
IMMUNOTECH, Marseille, France 3 CD41 PI1.64 Kaplan, 5th
International Workshop on Human Leukocyte 3 Differentiation
Antigens CD42a Bebl Becton Dickinson Immunocytometry, Mountain
View, Calif. 3 CD45 J33 IMMUNOTECH, Marseille, France 3 MEM28 Dr.
Vaclav Horejsi, Institute of Molecular 1 CD45RA 8D2.2 Craig et al.
1994, StemCell Technologies, Vancouver, Canada 1 L48 Becton
Dickinson Immunocytometry, Mountain View, Calif. 3 CD56 T199
IMMUNOTECH, Marseille, France 3 MY31 Becton Dickinson
Immunocytometry, Mountain View, Calif. 3 CD66e CLB/gran10 CLB,
Central Laboratory of the Netherlands, Red Cross Blood 3
Transfusion Service CD66b B13.9 CLB, Central Laboratory of the
Netherlands, Red Cross Blood 3 Transfusion Service 80H3 IMMUNOTECH,
Marseille, France 3 *preferred antibody based on performance in
magnetic cell separations
[0169]
3TABLE 3 Antibody Cocktails to Purify Specific Types of Lineage
Committed Cells Desired Cell type source of cells cocktail of
antibodies Monocytes Ficolled Blood anti-glycophorin A, anti-CD2,
CD3, CD56, CD19 Whole Blood anti-glycophorin A, anti-CD2, CD3,
CD56, CD19, CD66b B-Cells Ficolled Blood anti-glycophorin A,
anti-CD3, CD56, CD14, CD16, CD2 Whole Blood anti-glycophorin A,
anti-CD3, CD56, CD14, CD66b, CD16, CD2 T-Cells Ficolled Blood
anti-glycophorin A, anti-CD19, CD56, CD16, CD14 Whole Blood
anti-glycophorin A, anti-CD19, CD56, CD66b, CD16, CD14 CD4+ T-Cells
Ficolled Blood anti-glycophorin A, anti-CD19, CD56, CD8, CD16, CD14
Whole Blood anti-glycophorin A, anti-CD19, CD56, CD8, CD66b, CD16,
CD14 CD8+ T-Cells Ficolled Blood anti-glycophorin A, anti-CD19,
CD56, CD4, CD16, CD14 Whole Blood anti-glycophorin A, anti-CD19,
CD56, CD4, CD66b, CD16, CD14 NK Cells Ficolled Blood
anti-glycophorin A, anti-CD19, CD3, CD14, CD4 Whole Blood
anti-glycophorin A, anti-CD19, CD3, CD66b, CD14, CD4 Basophils
Whole Blood anti-glycophorin A, anti-CD2, anti-CD3, anti-CD14,
anti-CD15, anti-CD16, anti-CD19, anti-CD24, anti-CD34, anti-CD36,
anti-CD56 and anti-CD45RA Dendritic Cells Whole Blood
anti-glycophorin A, anti-CD3, anti-CD14, anti-CD16, anti-CD19,
anti-CD34, anti-CD56 and anti-CD66b Granulocytes Whole Blood
anti-glycophorin A, anti-CD2, anti-CD56, anti-CD19, anti-CD14, and
anti-CD3
[0170]
4TABLE 4 Antibodies Recognizing Non-Hematopoietic Antigens
Expressed on Epithelial Tumor Cells. Disease Antibody Antigen
Supplier/Developer Breast and Lung 5E11 unknown, breast carcinoma
STI Carcinoma 6E7 unknown, breast carcinoma STI H23A unknown,
breast carcinoma ATCC RAR9941 epithelial glycoprotein Baxter,
Germany RAR9948 epithelial glycoprotein Baxter, Germany RAR9938
crb2 Baxter, Germany C13B5 crb2 Immunotech, Marseille, France BRST
1 BCA 225 ID Labs BRST 3 TAG-72 ID Labs CA15.3 MAM-6, mucin ID Labs
CA27.29 MAM-6, mucin Cedarlane BerEp4 HEA DAKO Neuroblastoma UJ13A
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
[0171]
5TABLE 5 Purity and Yield of Human CD34.sup.+ CD38.sup.- Cells
Obtained Using the Primitive Progenitor Enrichment Procedure %
CD34.sup.+ % CD34.sup.+ % Yield CD38.sup.- CD38.sup.- CD34.sup.+
Start Enriched CD38.sup.- Cell Sample n Fraction Fraction Cells
Mobilized PB 3 0.02 .+-. 0.01 67 .+-. 6 50 .+-. 5 Frozen CB 1 0.16
78 20 BM 3 0.03 .+-. 0.01 61 .+-. 11 80 .+-. 10 Frozen BM 6 0.05
.+-. 0.01 34 .+-. 6 90 .+-. 20 %CD34+ CD38- cells in start fraction
are typically too low to detect accurately, therefore values given
are rough estimates. Accordingly, % recovery values, which
represent the present ratio of input vs. recovered absolute numbers
of % CD34+ CD38*-cells, are also relatively inaccurate.
[0172]
6TABLE 6 Enrichment and Yield of Long-Term Culture Initiating Cells
(LTC-IC) Using the Primitive Progenitor Enrichment Procedure Type
of Cells % Yield of LTC-IC Fold-Enrichment of LTC-IC Mobilized PB
160 2,000 70 1,000 Frozen CB 180 4,000 BM 110 10,000 110 5,500
[0173]
7TABLE 7 Purging Breast Carcinoma Cells (BT20 or T47D cells).
Anti-Breast Carcinoma Log Tumor Cell Cell Type Lineage Depletion
Antibodies Depletion Previously Frozen Bone Purge Only 5E11 1.8
Marrow 5E11, H23A 3.7, 3.7 5E11, 6E7 3.0 Previously Frozen Bone
Lineage Depletion and Purge 5E11 >5.8, 3.9, 4.7 Marrow RAR
>5.8, 4.3, 4.7 BRST1 4.9 5E11, H23A >5.2, 4.4 5E11, RAR,
BRST1 >5.8 Peripheral Blood Purge only 5E11 1.9, 1.9
Leukapheresis H23A 1.7 5E11, H23A 2.3 Peripheral Blood Lineage
Depletion and Purge 5E11, H23A 5.6 Leukapheresis Fresh Bone Marrow
Lineage Depletion and Purge 5E11, H23A 4.6, 4.4
[0174]
8TABLE 8 Recovery of Hematopoietic Colony Forming Cells During
Lineage Depletion To Enrich For Progenitors % Recovery Colony Assay
mean range CFU-GM 60-100 75 BFU-E 71-100 92 LTCIC 72->100
100
[0175]
9TABLE 9 Enrichment of CAMA Breast Carcinoma Tumor Cells From Bone
Marrow # CAMA in % CAMA in % CAMA in % Recovery Log Enrich. Exp #
Sample Start Start Flow CAMA CAMA 1 BM 1.1/10.sup.2 1.06 91.07
72.41 1.9 2 BM 2.2/10.sup.2 2.18 96.40 44.12 1.6 2.1/10.sup.3 0.21
82.16 75.00 2.6 2.1/10.sup.4 0.02 32.01 60.00 3.2 3 BM 2.6/10.sup.3
0.26 62.54 * 2.4 2.6/10.sup.4 0.026 11.21 * 2.6 2.6/10.sup.5 0.0026
2.01 * 2.9 2.6/10.sup.6 0.00026 0.13 * 2.7 *Cell numbers were too
low to count accurately.
[0176]
10TABLE 10 Purity, Recovery, and Enrichment of CAMA Breast
Carcinoma Tumor Cells Seeded into Previously Frozen Peripheral
Blood Mononuclear Cells Start Enriched Fraction % Purity % Purity %
Recovery Log Enrichment 0.3 95.5 9.0 2.5 0.03 65.3 13.3 3.4 0.003
17.1 12.1 3.8 0.3 96.7 14.3 2.5 0.03 52.4 13.1 3.3 0.003 82.7 14.8
4.5 0.3 92.7 25.8 2.5 0.03 61.4 17.0 3.3 0.003 16.8 7.0 3.7 0.3
91.2 14.5 2.5 0.03 61.3 17.9 3.3 0.003 18.0 8.0 3.8 0.02 24.5 47.2
3.1 0.02 9.3 42.9 2.7 0.1 97.7 85.8 2.9 0.01 81.0 86.1 3.9 0.001
21.4 62.1 4.3 0.004 6.6 4.4 3.2 0.03 42.8 12.5 3.2 0.02 35.7 12.8
3.3 0.01 40.4 62.3 3.5 0.01 36.3 54.2 3.4 0.01 33.7 53.0 3.4 0.02
43.3 25.2 3.4 0.02 52.9 38.1 3.5 0.02 26.9 113.0 3.2 0.02 34.7 71.9
3.3
[0177]
11TABLE 11 Purity and Enrichment of CAMA Breast Carcinoma Tumor
Cells Seeded into Previously Frozen Peripheral Blood Mononuclear
Cells: Antibody Composition of the Invention vs. CD45 Depletion
Only Start Enriched Fraction Cocktail % Purity % Purity Log
Enrichment Antibody Cocktail 0.001 21.4 4.3 Anti-CD45 only 0.001
6.2 3.8 Antibody Cocktail 0.03 42.8 3.2 Anti-CD45 only 0.03 6.6 2.4
Antibody Cocktail 0.02 35.7 3.3 Anti-CD45 only 0.02 5.4 2.4
Antibody Cocktail 0.01 40.4 3.5 Anti-CD45 only 0.01 11.8 3.0
Antibody Cocktail 0.01 36.3 3.4 Antibody Cocktail 0.01 33.7 3.4
Anti-CD45 only 0.01 8.0 2.8 Antibody Cocktail 0.02 43.3 3.4
Anti-CD45 only 0.02 20.1 3.1 Antibody Cocktail 0.02 26.9 3.2
Antibody Cocktail 0.02 34.7 3.3 Anti-CD45 only 0.02 3.5 2.3
[0178]
12TABLE 12 Purity, Recovery, and Enrichment of Cytokeratin+ Cells
from Pleural Effusions Start Enriched Fraction Sample # % Purity %
Purity % Recovery Log Enrichment 1 0.3 85.9 22.5 2.5 2 1.5 84.0
10.0 1.8 3 4.4 56.2 24.8 1.1 4 2.9 94.4 14.9 1.5 5 2.8 87.7 21.2
1.5 6 19.5 35.4 67.5 0.3 7 17.4 99.6 11.7 0.8 8 0.3 57.6 6.9 2.2 9
2.1 93.4 9.2 1.6 10 0.3 16.7 0.5 1.8 11 3.7 91.8 7.6 1.4 12 2.3
86.5 61.7 1.6 13 0.0 3.6 14 12.3 82.2 14.8 0.8 15 74.6 94.5 8.6
0.1
[0179]
13TABLE 13 Purity, Recovery, and Enrichment of Cytokeratin+ Cells
from Pleural Effusions Seeded into Previously Frozen Peripheral
Blood Mononuclear Cells Start Enriched Fraction Frequency % Purity
% Purity % Recovery Log Enrichment 3/10.sup.5 0.003 16.3 44.6 3.8
3/10.sup.6 0.0003 2.4 42.3 3.9 9/10.sup.6 0.0009 12.6 28.0 4.2
9/10.sup.7 0.00009 0.8 8.6 3.9 1/10.sup.5 0.001 10.2 19.4 4.0
1/10.sup.6 0.0001 1.4 31.6 4.2 2/10.sup.5 0.0002 1.7 44.7 4.0
2/10.sup.7 0.00002 1.1 95.1 4.8 2/10.sup.7 0.00002 1.3 31.3 4.9
* * * * *