U.S. patent application number 10/051751 was filed with the patent office on 2002-11-28 for novel antibody composition for isolating human cells from human-murine chimeric hematopoietic cell suspensions.
This patent application is currently assigned to STEMCELL TECHNOLOGIES INC.. Invention is credited to Eaves, Connie J., Thomas, Terry E..
Application Number | 20020177176 10/051751 |
Document ID | / |
Family ID | 31186110 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177176 |
Kind Code |
A1 |
Thomas, Terry E. ; et
al. |
November 28, 2002 |
Novel antibody composition for isolating human cells from
human-murine chimeric hematopoietic cell suspensions
Abstract
The present invention relates to an antibody composition which
contains antibodies directed to murine leukocyte and murine
erythroid cells. This composition is used in a novel negative
selection process to enrich for human hematopoietic cells from a
sample from human-murine chimeric mice. The invention also relates
to kits for carrying out this process.
Inventors: |
Thomas, Terry E.;
(Vancouver, CA) ; Eaves, Connie J.; (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: |
31186110 |
Appl. No.: |
10/051751 |
Filed: |
May 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10051751 |
May 20, 2002 |
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09363677 |
Jul 30, 1999 |
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6342344 |
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60094844 |
Jul 31, 1998 |
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Current U.S.
Class: |
435/7.21 ;
435/372; 435/7.5 |
Current CPC
Class: |
G01N 33/56966 20130101;
C07K 16/28 20130101; G01N 33/5094 20130101; G01N 2333/70539
20130101; G01N 2333/70589 20130101 |
Class at
Publication: |
435/7.21 ;
435/7.5; 435/372 |
International
Class: |
G01N 033/567; G01N
033/53; C12N 005/08 |
Claims
We claim:
1. A negative selection process for enriching and recovering human
cells in a sample containing human cells and murine cells
comprising: (a) reacting the sample with an antibody composition
containing antibodies capable of binding to murine leukocytes under
conditions such that conjugates are formed between the antibodies
and murine leukocytes; (b) removing the conjugates; and (c)
recovering a cell population which is enriched in human cells and
depleted of murine leukocytes.
2. A process according to claim 1 further comprising adding
antibodies capable of binding to murine erythroid cells in step (a)
and recovering a cell population which is enriched in human cells
and depleted of murine hematopoietic cells in step (c).
3. A process according to claim 1 wherein the antibody capable of
binding to murine leukocytes is a murine anti-CD45 antibody.
4. A process according to claim 2 wherein the antibody capable of
binding to murine erythroid cells is TER119.
5. A process according to claim 2 wherein the antibodies are
monoclonal antibodies.
6. A process as claimed in claim 5 wherein the antibodies are
labelled with a marker or they are conjugated to a matrix.
7. A process as claimed in claim 5 wherein the antibodies are
labelled with biotin or fluorochrome.
8. A process as claimed in claim 6 wherein the matrix is magnetic
beads, a panning surface, dense particles for density
centrifugation, an adsorption column, or an adsorption
membrane.
9. A process as claimed in claim 8, wherein each of the monoclonal
antibodies to the murine leukocytes and murine erythroid cells is
incorporated in a tetrameric antibody complex wherein each
tetrameric antibody complex comprises a first monoclonal antibody
of a first animal species can bind either leukocytes or erythroid
cells, 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.
10. A process as claimed in claim 9 wherein each of the monoclonal
antibodies is biotinylated and coupled to a separation matrix via a
tetrameric antibody complex which recognises biotin and the
matrix.
11. An antibody composition comprising an antibody specific for
murine leukocytes and an antibody specific for murein erythroid
cells.
12. An antibody composition according to claim 11 wherein the
antibody specific for the murine leukocytes is murine
anti-CD45.
13. An antibody composition according to claim 11 wherein the
antibody specific for the murine leukocytes is an anti-MHC-I
antibody.
14. An antibody composition according to claim 11 wherein the
antibody capable of binding the murine erythroid cells is TER119.
Description
[0001] This application claims benefit from U.S. provisional
application serial No. 60/094,844 filed on Jul. 31, 1998 which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention provides novel antibody compositions
and negative selection processes for enriching human cells from
murine/human chimeric haematopoietic cell suspension.
BACKGROUND OF THE INVENTION
[0003] Human cells are often transplanted into mice in order to
study various diseases as the study of human cells in such diseases
cannot always be adequately modelled in vitro.
[0004] Human/Murine transplant chimeras have been used to study
human autoimmune diseases, such as Graves disease (Yoshikawa, N. et
al., 1997; Weetman, A. P., 1996). Chimeric mice have also been used
to evaluate the efficacy of anti-viral agents in the treatment of
human immunodeficiency virus (HIV) and Epstein Barr Virus (EBV)
(Jenkins M. et al., 1998; Fuzzati-Armentero, M. T., 1998). However,
in such models it has been difficult to efficiently retrieve human
cells from the chimeric mice to enable further assays.
[0005] Human/Murine chimeric mice are also used to study
hematopoietic stem cells. The hematopoietic stem cell is identified
by its distinct functional capabilities, including self-renewal and
long-term repopulation of all hematopoietic lineages. In vitro
assays, such as long-term culture--initiating cells (LTC-IC)
(Sutherland, H. J., et al., 1989) are not entirely predictive of
repopulating and homing potential in vivo and therefore, several
groups have transplanted human hematopoietic stem cells into
RAG-/-(Koyanagi, Y. et al., 1997) or severe combined immune
deficiency (SCID) mice (McCune, J. M. et al., 1988; Kamel-Reid S.
and Dick J. E., 1988; Kyoizumi S. et al., 1992; Larochelle, A., et
al., 1996); or non obese diabetic SCID (NOD.SCID) (Cashman, J. D.,
et al. 1997). The surviving transplanted human cells and their
progeny may be very rare in bone marrow and blood. This creates
difficulties in determining the success of engraftment and makes
further functional assay of the surviving engrafted human cells not
feasible. Therefore, there is a need to develop a method to enrich
for human cells allowing the detection and isolation of these low
frequency human cells in chimeric SCID/Hu, NOD.SCID/Hu or RAG-/-/Hu
mice.
SUMMARY OF THE INVENTION
[0006] The present inventors have developed an antibody composition
for use in enriching human cells from human-murine chimeric
hematopoietic cell suspensions. The antibodies in the antibody
composition are specific for selective markers associated with
murine cells.
[0007] In particular, the present inventors have found that a
negative selection technique using an antibody composition
containing an antibody specific for murine leukocytes (such as
anti-CD45 and/or anti major histocompatibility complex class I
(MHC-I)) alone or in combination with an antibody capable of
binding to murine erythroid cells gives a cell preparation highly
enriched for human cells. Accordingly, the present invention
provides an antibody composition comprising an antibody specific
for murine leukocytes and an antibody specific for murine erythroid
cells.
[0008] Preferably, the present invention provides an antibody
composition comprising an antibody specific for murine CD45 and an
antibody capable of binding to a murine erythroid cells. CD45 is a
pan-leukocyte maker expressed on all hematopoietic cells with the
exception of erythroid cells (Ledabetter, J. A and Herzenburg, L
A., Immunol. Rev. 47:63 (1979); Thomas, M. L. Ann. Rev. Immunol.
7:339-369 (1989); Van Ewijk, W. et al., J. Immnumol. 127:2594
(1981)). Human cells will be enriched from human-murine chimeric
hematopoietic cell suspension by depleting only CD45.sup.+ murine
cells although the presence of murine erythroid cells will limit
the degree of enrichment. Combining an antibody which recognizes
murine erythroid cells with an anti-murine CD45 antibody will
deplete all murine hematopoietic cells and thereby achieve
extensive enrichment of human cells thus enabling detection and
further study.
[0009] The enrichment and recovery of human cells using the
antibody composition of the invention in a negative selection
technique has many advantages over conventional positive selection
techniques. Most importantly, the recovered human cells are not
labelled or coated with antibodies thereby making them highly
suitable for further study.
[0010] The present invention includes a negative selection process
for enriching and recovering human cells in a sample containing
human cells and murine cells comprising:
[0011] (a) reacting the sample with an antibody composition
containing antibodies capable of binding to murine leukocytes under
conditions such that conjugates are formed between the antibodies
and murine leukocytes;
[0012] (b) removing the conjugates; and
[0013] (c) recovering a cell population which is enriched in human
cells and depleted of murine leukocytes.
[0014] In a preferred embodiment, the present invention
provides
[0015] (a) reacting the sample with an antibody composition
containing antibodies capable of binding to murine leukocytes and
antibodies capable of binding to murine erythroid cells under
conditions such that conjugates are formed between the antibodies
and the murine leukocytes and murine erythroid cells;
[0016] (b) removing the conjugates; and
[0017] (c) recovering a cell population which is enriched in human
cells and depleted of murine leukocytes and murine erythroid
cells.
[0018] The present invention also includes a kit useful in
performing the process of the invention comprising antibodies
specific for murine leukocytes and murine erythroid cells and
instructions for performing the process of the invention.
[0019] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples while indicating preferred embodiments of the
invention are given by way of illustration only, since various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The invention will now be described in relation to the
drawings in which:
[0021] FIG. 1 is a schematic drawing of magnetic labelling of
murine cells.
[0022] FIG. 2 is a schematic drawing illustrating the enrichment
method the invention.
[0023] FIG. 3 is a FACS profile showing murine marrow cells seeded
with human cord blood cells (1:100) before (A and C) and after (B
and D) processing using the method of the invention.
[0024] FIG. 4 is a FACS profile showing cells from chimeric murine
bone marrow before (A) and after (B) depletion of murine cells
using the method of the invention.
[0025] FIG. 5 is a FACS profile of marrow cells from human/murine
(NOD/SCID) chimeric bone marrow before (pre-column) and, after
(post-column) depletion of murine cells using the method of the
invention. Human CD3.sup.+ and human CD34.sup.+ cells are
detected.
DETAILED DESCRIPTION OF THE INVENTION
[0026] I. Antibody Composition
[0027] As hereinbefore mentioned, the present invention relates to
an antibody composition comprising an antibody specific for murine
leukocytes in combination with an antibody specific for murine
erythroid cells. Preferably, the antibody specific for murine
leukocytes is anti-CD45 or anti-MHC-I and the antibody specific for
murine erythroid cells is TER119.
[0028] 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
recombinantly produced binding partners.
[0029] Polyclonal antibodies against selected antigens on the
surface of murine 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 or rats.
[0030] Preferably, monoclonal antibodies are used in the antibody
compositions of the invention. Monoclonal antibodies specific for
selected antigens on the surface of murine cells may be readily
generated using conventional techniques (see U.S. Pat. Nos. RE
32,011, 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, which are also incorporated herein by reference).
[0031] Other techniques may also be utilized to construct
monoclonal antibodies (see William D. Huse et al., "Generation of a
Large Combinational Library of the Immunoglobulin Repertoire in
Phage Lambda," Science 246:1275-1281, December 1989; see also L.
Sastry et al., "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, August 1989; see
also Michelle Alting-Mees et al., "Monoclonal Antibody Expression
Libraries: A Rapid Alternative to Hybridomas," Strategies in
Molecular Biology 3:1-9, January 1990; these references describe a
commercial system available from Stratacyte, La Jolla, Calif.,
which enables the production of antibodies through recombinant
techniques).
[0032] Similarly, binding partners may be constructed utilizing
recombinant DNA techniques. Within one embodiment, the genes which
encode the variable region from a hybridoma producing a monoclonal
antibody of interest are amplified using nucleotide primers for the
variable region. These primers may be synthesized by one of
ordinary skill in the art, or may be purchased from commercially
available sources. The primers may be utilized to amplify heavy or
light chain variable regions, which may then be inserted into
vectors such as ImmunoZAP.TM. H or ImmunoZAP.TM. L (Stratacyte),
respectively. These vectors may then be introduced into E. coli for
expression. Utilizing these techniques, large amounts of a
single-chain protein containing a fusion of the V.sub.H and V.sub.L
domains may be produced (See Bird et al., Science 242:423-426,
1988). In addition, such techniques may be utilized to change a
"murine" antibody to a "human" antibody, without altering the
binding specificity of the antibody.
[0033] Antibodies against selected antigens on the surface of
murine cells may also be obtained from commercial sources. In this
regard, antibodies against murine CD45 include Pharmingen clone
30-F11, Caltag clone YW62.3, Serotec clone YW62-3 and Sigma clone
13-2. Antibodies to murine MHC-I include Deveron clone KDH3, UMRD
Inc. clone H58A, Bachem Bioscience clones ERHR52 and ERMPH2.
Antibodies against murine erythroid cells include Pharmingen clone
TER119 (Ikuta, K. et al., Cell 62:863-874; Ogawa, M. et al. J. Exp.
Med. 174:63-71). The anti-murine CD45 antibody depletes CD45
positive murine cells. Tests have shown that the above clones
30-F11 and TER119 do not bind human cells. The TER119 antibody
recognizes murine erythroid cells.
[0034] II. Method of the Invention
[0035] The present invention includes a negative selection process
for enriching and recovering human cells in a sample containing
human cells and murine cells comprising:
[0036] (a) reacting the sample with an antibody composition
containing antibodies capable of binding to murine leukocytes under
conditions such that conjugates are formed between the antibodies
and murine leukocytes;
[0037] (b) removing the conjugates; and
[0038] (c) recovering a cell population which is enriched in human
cells and depleted of murine leukocytes.
[0039] In a preferred embodiment, the present invention
provides
[0040] (a) reacting the sample with an antibody composition
containing antibodies capable of binding to murine leukocytes and
antibodies capable of binding to murine erythroid cells under
conditions such that conjugates are formed between the antibodies
and the murine leukocytes and murine erythroid cells;
[0041] (b) removing the conjugates; and
[0042] (c) recovering a cell population which is enriched in human
cells and depleted of murine leukocytes and murine erythroid
cells.
[0043] The sample can be any sample from a human/murine chimeric
mouse including, but not limited to, blood, bone marrow, spleen,
thymus, liver and lymph node samples.
[0044] Conditions which permit the formation of cell 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 murine cells in the sample.
[0045] The antibodies in the antibody composition 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 or anti-biotin
antibody 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 (Kemshead 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.), adsorption columns (Berenson et al.
1986, Journal of Immunological Methods 91:11-19.), and adsorption
membranes (Nordon et al. 1994). The antibodies may also be joined
to a cytotoxic agent such as complement or a cytotoxin, to lyse or
kill the targeted murine cells.
[0046] The antibodies in the antibody composition may be directly
or indirectly coupled to a matrix. For example, the antibodies in
the composition of the invention may be chemically bound to the
surface of magnetic particles using, for example 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 murine cells, and the
murine cells having the antigens on their surfaces.
[0047] 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.
[0048] The antibodies in the antibody composition 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.
[0049] Bispecific antibodies contain a variable region of an
antibody in the 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.
[0050] 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 antibodies are from a first animal species.
The first and second antibodies 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 antibodies 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).
[0051] 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.
[0052] 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 Kemshead 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).
[0053] FIG. 1 is a schematic representation of magnetic cell
labelling using biotinylated antibodies, tetrameric antibody
complexes and colloidal dextran iron.
[0054] In accordance with the magnetic separation method, the
sample containing the human cells to be recovered, is reacted with
the above described antibody reagents, preferably tetrameric
antibody complexes and biotinylated anti-mouse antibodies, so that
the antibody reagents bind to the targeted murine cells present in
the sample to form cell conjugates of the targeted murine cells and
the antibody reagents. Cells are first labelled with biotinylated
anti-CD45 and TER119, washed and then incubated with tetrameric
antibody complexes followed by magnetic colloid. The reaction
conditions are selected to provide the desired level of binding of
the targeted murine cells and the antibody reagents. Preferably the
sample is incubated with the antibody reagents for a period of 5 to
60 minutes at either 4-8.degree. C. or ambient room temperature.
The concentration of the antibody reagents are selected to optimize
cell labelling in a sample of 2-8.times.10.sup.7 in nucleated cells
per ml. 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 although the time may be longer or
shorter depending upon condition chosen. 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.
[0055] 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 permanent gap magnet with a 0.5-2.0 inch gap 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.
[0056] 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 LTC-IC in culture may also be assessed. The efficiency of
the separation procedure may also be determined by monitoring the
recovery of human CD.sup.45+ cells.
[0057] III. Uses of the Composition and Processes of the
Invention
[0058] The composition and processes of the invention may be used
in the processing of samples including marrow and spleen from
human/murine chimeric mice. Using the processes of the invention it
is possible to recover a highly purified preparation of human cells
from the human/murine chimeric mice for the purpose of studying,
human stem cells; human hematopoietic disease; human immune
function, development and pathophysiology; human responses to
infection; and transplantation biology.
[0059] The present invention also includes a kit useful in
performing the process of the invention comprising antibodies
specific for murine leukocytes and murine erythroid cells and
instructions for performing the process of the invention.
[0060] The following non-limiting examples are provided for
illustration of the present invention.
EXAMPLES
Example 1
[0061] Methodology
[0062] Human cells may be recovered from chimeric mouse marrow or
spleen using the antibody composition of the invention containing
biotinylated antibodies (anti-CD45 and TER119) directed against all
murine hematopoietic cells. The labelled cells are linked to
magnetic dextran-iron particles via bispecific anti-biotin
anti-dextran tetrameric antibody complexes. FIG. 1 illustrates the
indirect magnetic labelling of murine cells. Cells are first
labelled with a cocktail of biotinylated antibodies directed
against murine cell surface markers. Murine cells are then
cross-linked to magnetic dextran iron particles using tetrameric
antibody complexes comprised of two murine IgG.sub.1 monoclonal
antibodies held in tetrameric array by two rat anti-mouse IgG.sub.1
monoclonal antibody molecules. One murine antibody molecule
recognizes biotin and the other recognizes dextran on the magnetic
particle. The immunomagnetically labelled murine cells are then
removed from the cell suspension in a magnetic column leaving a
cell suspension enriched for unlabelled human hematopoietic cells.
The magnetically labelled murine cells bind to the column.
[0063] Flow Cytometry
[0064] Human cells were detected by FACS analysis following
staining with a combination of FITC labelled anti-human CD45
(leukocytes) antibody and PE labelled anti-human CD71
(erythrocytes) antibody. Murine cells were detected with a
combination of anti-murine CD45 antibody and TER119 antibody. Human
subsets were detected by double staining with the appropriate FITC
labelled anti-human antibodies (anti CD3 and anti CD34).
[0065] Chimeric Mice
[0066] The SCID mutation or RAG knockout results in failure of the
VDJ joining mechanism during rearrangement of antigen receptor
genes. Consequently, RAG knockout and CB-17 mice homozygous for the
SCID mutation lack functional B and T cells (Bosma, G. C., Nature,
301:527, 1983; Bosma, M J. and Carroll, A. M., Ann. Rev. Immunol.,
9:323, 1991; Koyanagi, Y. et al., Leukemia, 11:109-112, 1997). SCID
and RAG.sup.-/- mice do, however, possess normal NK and myeloid
function which can limit the survival of cells transplanted into
these mice (Mosier, D. E. et al., Nature, 335:256, 1988; McCune, J.
M., et al., Science, 241:1632, 1988). The SCID mutation was
therefore back-crossed onto the NOD/Lt mouse, which is defective in
innate immune function. The resultant NOD/SCID mouse is defective
in both lymphoid and myeloid function and will readily accept the
long-term survival of human hematopoietic cells (Shultz, L. D. et
al., J. Immunol., 154:180, 1995).
[0067] Results
[0068] Verification of Specificity
[0069] Normal human cord blood mononuclear cells (HCMC) were seeded
into NOD/SCID bone marrow suspensions at human:murine cell ratios
of 1:1, 1:10 or 1:100. Human cells were then recovered from the
mixture using the method of the invention and detected by FACS as
shown in FIG. 3. FACS plots show staining of 1:100 mixtures of
human cells and mouse marrow before (A and C) and following (B and
D) enrichment of human cells. Cells were stained with anti-murine
CD45 and TER119 (A and B) or with anti-human CD45 and CD71 (C and
D). The recovery of various types of human and murine cells is
given in Table 1.
[0070] Isolation of Human Cells from Transplanted Mice
[0071] NOD/SCID mice were irradiated with a sub-lethal dose of
radiation prior to intravenous injection of 2.times.10.sup.7 to
2.times.10.sup.8 HCMC (equivalent to 0.7 to 3.times.10.sup.6
CD34.sup.+ cells). Six weeks later NOD/SCID bone marrow was
harvested and human cells were detected by FACS. Without enrichment
of human cells, mice transplanted with higher doses
(3.times.10.sup.6) of CD34.sup.+ cells were found to be
successfully engrafted with human cells. In contrast, mice
transplanted with lower doses (0.7.times.10.sup.6) of CD34.sup.+
cells were minimally positive or negative for human cells.
Following depletion of murine cells using the method of the
invention, human cells were clearly detected by FACS in the marrow
of these low dose CD34 mice.
[0072] FIG. 4 and Table 2 show the enrichment and recovery of
CD45.sup.+ human cells from chimeric mouse bone marrow using the
method of the invention. FIG. 4 illustrates the low level of
engraftment of human cells in these chimeric mice which was barely
detectable prior to enrichment (A) was the vast majority of events
following enrichment of human cells using the method of the
invention (B). Cells were stained with anti-human CD45 and
anti-CD71. Table 3 shows the percent recovery of various
populations of human and murine cells using the method of the
invention to enrich for human cells.
[0073] Detection and Isolation of Rare Human Hematopoietic Cell
Subsets
[0074] The enrichment of subpopulates of human cells in SCID/Hu
bone marrow is often too low to detect or sort using FACS.
Following enrichment using the method of the invention, minor
populations of human cells were detected by FACS. The inventors
demonstrated the presence of CD3.sup.+ and CD34.sup.+ human cells
in the marrow of engrafted mice. These populations were not
detectable by FACS prior to enrichment (FIG. 5).
[0075] Enrichment of Human Cells for Functional Studies
[0076] Typically 10.sup.9 SCID/Hu bone marrow cells are required to
produce the minimal number of human cells needed in functional
assays. This is beyond the capacity of most FAC sorters but can be
easily accommodated with the method of the invention. The marrows
from several SCID/Hu mice are pooled and using the method of the
invention, sufficient unlabelled human cells were obtained to
perform colony assays in methylcellulose. The resultant colonies
were found to be exclusively human using standard cytogenetic
techniques. Table 4 shows the human colony forming cell (CFC) assay
results for the bone marrow of four individual chimeric mice. Prior
to processing none of the bone marrow suspensions had sufficient
human cells to plate in the CFC assay. Following enrichment of
human cells with the method of the invention human colonies were
detected in all four samples.
[0077] Conclusions
[0078] The antibody composition of the invention was able to
specifically deplete murine cells providing a suspension of viable
human cells in sufficient numbers to perform functional and
phenotypic analyses. This facilitated detection of human cells in
mice and hence assessment of engraftment. Enrichment of human cells
also allowed the detection and isolation of rare subsets of human
cells.
[0079] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety.
[0080] 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.
[0081] Detailed References
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1TABLE 1 Recovery of Various Types of Murine and Human Cells after
Immunomagnetic Depletion of the Murine Cells from Artificial
Mixtures of Human Cord Blood and Murine Bone Marrow Cells Percent
Recovery Mixtures Mixtures Mixtures with 50% with 10% with 1%
Populations Analyzed Human Human Human Genotype Phenotype Cells
Cells Cells Human CD45/71.sup.+ 84 .+-. 14 61 .+-. 13 58 .+-. 5
CD34.sup.-CD19/20.sup.+ 67 .+-. 0 48 .+-. 3 75 .+-. 16
CD45/71.sup.+/CD15.sup.+ 99 50 .+-. 11 56 .+-. 7 CD34.sup.+ 74 .+-.
6 90 .+-. 19 140 .+-. 40 Mouse CD45.sup.+ <0.2 <0.05 <0.03
Ter-119.sup.+ 0.04 <0.06 <0.03 Values shown are the mean .+-.
SEM of values from 4 experiments in 2 of which a 50% mixture was
not included. Where no errors are shown, only a single measurement
was made. In many of the experiments, murine CD45.sup.+ or
Ter-119.sup.+ cells, if present, were below the threshold set for
this assay (<5/5000 positive events over background). In these
instances, a number equivalent to the threshold value was used to
calculate upper limits (designated as < values).
[0104]
2TABLE 2 The recovery of human CD45.sup.+ cells from chimeric mouse
marrow following enrichment of human cells using the method of the
invention. % Human CD45.sup.+ Cells % Recovery Pre-Column
Post-Column Human CD45.sup.+ Cells Mouse 1 4.2 98.0 58 Mouse 2 64.9
98.0 61 Mouse 3 3.6 99.5 86 Mouse 4 8.8 98.5 94 Mouse 5 20.6 98.9
43 Mouse 6 48.0 99.4 85
[0105]
3TABLE 3 Percent Recovery of Various Populations of Human and Mouse
Cells During Depletion of Mouse Cells from Marrow Cells Harvested
from NOD/SCID Mice Engrafted with Human Cells. Populations Analyzed
Percent Recovery Geonotype Phenotype Experiment 1 Experiment 2
Human CD45.sup.+/71.sup.+ 59 .+-. 6 94 .+-. 14
CD34.sup.-CD19.sup.+/20.sup.+ 68 .+-. 9 100 .+-. 10
CD45.sup.+/71.sup.+CD15.sup.+ 29 .+-. 5 57 .+-. 18 CD34.sup.+ 45
.+-. 5 97 .+-. 19 CFC 47 .+-. 15 ND Mouse CD45.sup.+ <0.08
<0.1 Ter-119.sup.+ <0.2 <0.1 Values shown are the mean
.+-. SEM of values for marrows harvested and processed individually
from 4 and 6 mice per experiment (no. 1 and 2, respectively). Zero
values (from mouse cell analyses) were treated as described in the
footnote to Table 1. ND = Note done.
[0106]
4TABLE 4 The detection of human colony forming cells (CFC) in the
marrow of chimeric mice following enrichment of human cells
(post-column) using the method of the invention. Total Colonies
Counted Pre-Column Post-Column Cells Plated Cytogenics Mouse 1 ND
13 5 .times. 10.sup.4 Hu Mouse 2 ND 32 10.sup.4 Hu Mouse 3 ND 113
10.sup.4 Hu Mouse 4 ND 149 10.sup.4 Hu ND: Insufficient human cells
are present in the pre-column samples to allow plating in CFC
assays
* * * * *