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TECHNICAL FIELD
This invention relates to methods of immunoselection employing
avidin-biotin.
BACKGROUND OF THE INVENTION
Immunoselection is a generic term that encompasses a variety of techniques
in which the specificity of a selection system is conferred by an antibody
or an antibody-like molecule such as a lectin or hapten. An example of
such specificity is the affinity of an antibody for a specific cell
surface antigen. Two general types of immunoselection techniques are
practiced. Negative immunoselection involves the elimination of a specific
subpopulation of components from a heterogeneous population such as a
suspension of various cell types. Exclusively negative selection
techniques have an inherent disadvantage: Although specific component
types can be removed, the remaining components are an enriched but not a
pure population. In contrast, positive immunoselection refers to the
direct selection and recovery of a specific component, such as cells which
express a given specificity from among a heterogeneous group of
contaminating cell types. Rather than eliminating the undesired elements,
positive immunoselection techniques can lead directly to the selective
enrichment and even purification of targeted antigen-bearing cells.
Another difference between the two techniques is that viable, functional
cells are typically the desired end product of positive immunoselection
techniques, while the continuing viability of negatively selected cells is
usually not required.
Bone marrow transplantation provides an illustrative example of a clinical
application in which immunoselection techniques have shown promise. Bone
marrow transplantation is being utilized for treatment of increasing
numbers of patients with hematological malignancies, aplastic anemia,
solid tumors, certain congenital hematological disorders, and
immunodeficiency states. The basic aim of the bone marrow transplantation
treatment is to replace a defective component of the blood or immune
system by first destroying the defective cells in the patient (recipient)
and then transplanting omnipotential, hematopoietic stem cells derived
from either the recipient (autologous transplant) or another donor
(allogeneic transplant). The transplanted hemato-poietic stem cells can
potentially differentiate into normal cell types that will replace the
defective cells. Unfortunately, about seventy percent of patients with
aplastic anemia and even fewer with leukemia survive more than one year
following the bone marrow transplantation procedure. The major obstacles
that limit the success of this procedure include graft-versus-host disease
in patients given allogeneic transplants, and relapse possibly caused by
the presence of residual tumor cells following autologous marrow
transplants.
Investigators have demonstrated that graft-versus-host disease can be
prevented in animals by removing T cells from the donor marrow before
transplantation into the recipient. Similarly, successful autologous
marrow transplants have been performed in animals in which tumor cells
were first removed in vitro from the donor marrow before transplantation
into the recipient. The successes of such animal studies have stimulated
the development of various in vitro methods designed to eliminate T cells
or tumor cells from human bone marrow. These methods have included the use
of antibodies with complement, antibody-toxin conjugates, various physical
separation or fractionation procedures, chemotherapeutic agents, and
magnetic immunoselection. Another approach has been to use a
chromatography column made of gel substrate to which monoclonal antibodies
directed against T cells or tumor cells are directly linked.
Transplantation 38:136, 1984; Proceedings, American Society of Clinical
Oncology, Toronto 3:269 (Abstract #C-1052), 1984. Such a negative
immunoselection technique avoids the toxicity to hematopoietic stem cells
and decreased recovery of bone marrow that characterize some of the other
in vitro techniques. However, the large quantities of monoclonal
antibodies required for direct attachment to the gel (5 mg/ml) has made
scale up of that procedure for clinical application to human bone marrow
transplantation impractical.
The high binding efficiency between avidin and biotin has also been applied
in prior immunoselection techniques. Biotin is a vitamin present in minute
amounts in every living cell. Avidin is a glycoprotein isolated from raw
egg white. Biotin selectively combines with avidin with a remarkably high
affinity constant (K.sub.m =10.sup.-15 M). The avidin-biotin complex has
been used as a tool in molecular biology for the following objectives:
isolation of biotin-derivatized materials by affinity chromatography;
affinity labeling and identification studies; affinity cytochemical
labeling for localization studies in fluorescence and electron microscopy;
inhibition of bacteriophage; and study of cell surface molecular
interactions. Bayer and Wilcheck, TIBS, N257-N259, Nov. 1978. With regard
to the isolation of biotin-derivatized materials by affinity
chromatography, biotinized rat thymocytes were reportedly retrieved using
avidin which was covalently coupled to nylon meshes; no attempts were made
to remove the cells from the avidin. Exp. Cell Res. 100:213-217, 1976.
T-cells were reportedly depleted by treating spleen cells with
biotin-conjugated antibody directed to T-cell antigen, followed by panning
twice on avidin-coated plates. J.Exp.Med. 159:463-478, 1984. Another
negative immunoselection technique employing avidin-biotin is described in
U.S. Pat. No. 4,298,685.
Biotinylated antibodies have been used to label cells which were then
removed by passing them over immobilized avidin, but unfortunately the
order to use this technique for positive immunoselection, antibodies have
been conjugated with biotin analogs that reportedly have the advantage
that they adhere to avidin containing matrices but can be displaced by
washing with authentic biotin. J.Immunol.Meth. 56:269-280, 1983.
Biotinylated antibodies and avidin-coupled sheep erythrocytes were used for
rosette formation, followed by separating rosetting cells from
non-rosetting cells on a density gradient. This method was reported to
present the possibility of obtaining both positive and negative
subpopulations with high yield. J.Immunol.Meth. 67:389-394, 1984. However,
recovered cells are coated with sheep erythrocytes which could potentially
affect the function of the rosetted cell population.
U.S. Pat. Nos. 4,253,996 and 4,276,206 note that the application of
insolubilized avidin in the affinity chromatographic isolation of
biotin-containing molecules has been limited due to the problem in
recovery since the affinity of avidin to biotin is so high. Both patents
reportedly circumvent this problem by preparing avidin-Sepharose
conjugates with reduced affinity for biotin and thereby reportedly provide
an operable positive selection technique using the avidin-biotin system.
Uses of a second, anti-species antibody in conjunction with immunoassays
employing avidin and biotin are disclosed in U.S. Pat. Nos. 4,228,237,
4,468,470, and 4,496,654.
It would be advantageous to provide an improved negative immuno-selection
method whereby specific subpopulations of cells such as T cells and/or
tumor cells could be removed from heterogeneous cell populations such as
bone marrow cells with a high degree of selectivity and with a low
nonspecificity, using relatively small amount of antibody and minimal
manipulation of the nonselected cells. It would also be desirable to
provide an improved positive immunoselection system.
SUMMARY OF THE INVENTION
Pursuant to the invention, the selectivity of immunoselection systems
employing insolubilized avidin and biotinylated specific antibody is
amplified, and nonspecific recovery is improved, by employing a
biotin-conjugated antispecies immunoglobulin that is directed to one or
more non-biotinylated specific antibodies. In an illustrative embodiment,
a murine monoclonal antibody or antibody fragment is selected to bind to a
specific cell subpopulation. A heterogeneous suspension of cells that
includes the specific cell subpopulation is incubated with the specific
murine antibody. Unbound antibody is then washed away. The cell suspension
is then incubated with an antimouse immunoglobulin-biotin conjugate to
form a complex of antimouse-biotin/murine antibody on the cells of the
specific subpopulation. Excess antimouse-biotin is washed away. The cell
suspension is then passed over a column of insolubilized avidin, to which
cells of the targeted subpopulation are bound via avidin/biotin complexes.
Unadsorbed cells are washed from the column. The recovery of cell
populations that do not substantially react with nonbiotinylated specific
antibody is amplified using this method as compared with prior techniques
employing biotinylated specific antibody. Moreover, a plurality of
specific antibodies, e.g., a first antibody specific for one T cell
antigen and a second antibody specific for a second T cell antigen, can be
simultaneously incubated with a heterogeneous cell suspension without the
need to biotinylate each specific antibody. This embodiment has particular
advantages for removing and recovering certain cell populations from bone
marrow, providing excellent recovery of bone marrow and preservation of
hematopoietic stem cells for transplantation. Mixed populations of T cells
or of tumor cells can be conveniently and simultaneously removed with
minimal manipulation of the marrow cells. In related applications,
peripheral blood cells or tissue cell suspensions, e.g., spleen cells, can
be enriched by the removal of one or more specific cell types using this
method.
The invention also provides an improved positive immunoselection method:
After the aforesaid unadsorbed cells are washed from the column, the
remaining cells can be conveniently recovered by disassociating the
antispecies/specific antibody bond (which is much weaker than the
biotin/avidin bond). The recovered cells, e.g., hematopoietic stem cells
or activated killer cells, can be clinically employed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram of a preferred embodiment of the
immunoselection method of the present invention, showing both positive and
negative immunoselection techniques;
FIG. 2 is a graph showing the effect of the concentration of avidin linked
to Sepharose on the log removal of Daudi cancer cells and percent recovery
of noncancerous bone marrow cells after treatment with biotinylated
specific antibody 2H7, as described in Example 8;
FIG. 3 is a graph showing the effect of biotinylated anti-mouse antibody
B-GAMIg on log removal of Daudi cancer cells and percent recovery of
noncancerous bone marrow cells after treatment with nonbiotinylated murine
antibody 2H7, as described in Example 9;
FIG. 4 is a pair of graphs (A and B) showing the effect of biotinylated
anti-mouse antibody B-GAMIg on log removal of Daudi cancer cells and
percent recovery of noncancerous bone marrow cells after treatment with 10
ug/ml (A) or 30 gu/ml (B) of antibody DM1, as described in Example 11;
FIG. 5 is a fluorescence-activated cell sorter analysis (five panels) and
table demonstrating selective enrichment of an Ia-positive cell population
from a suspension of dog bone marrow mononuclear cells, as described in
Example 13;
FIG. 6 is a fluorescence-activated cell sorter analysis (four panels) and
table demonstrating selective enrichment of a T cell population from a
suspension of human bone marrow mononuclear cells, as described in Example
14;
FIG. 7 is a fluorescence-activated cell sorter analysis (three panels) and
table demonstrating selective enrichment of a T cell population from a
suspension of dog peripheral bone mononuclear cells, as described in
Example 16;
FIG. 8 is a schematic diagram of an on-line or off-line immunoselection
system for removing selected components from a patient's bloodstream; and,
FIG. 9 is a schematic cross section through an extracorporeal cartridge
that can serve as a dialyzer, cell collector, and bioreactor.
DESCRIPTION OF THE PREFERRED EMBODIMENT
Pursuant to the invention, the selectivity of selection systems employing
insolubilized avidin and biotinylated reagents is amplified, and
non-specific recovery is improved, by the use of an indirect sandwich
technique. In one embodiment an improved negative system is provided. A
suspension such as a cell suspension containing one or more targeted
components whose removal is desired is reacted with nonbiotinylated first
reagent(s) having binding specificity for the targeted component(s). A
targeted component can be, for example, a particular cell subpopulation,
and the first reagent a monoclonal antibody or antibody fragment directed
to antigens on the particular cells. The suspension can contain cells of
bone marrow, blood, lymph node, spleen, liver, or other tissues and
organs. The suspension and the first reagent are incubated to promote the
formation of complexes between the first reagent and the targeted
component, or first reagent/component complexes. Unreacted first reagent
is preferably then removed by, e.g., a washing step. The suspension
containing the first reagent/component complexes is next reacted with a
biotinylated second reagent, selected because it has specificity for the
first reagent, in order to associate or bind the first and second reagents
and thereby form biotinylated second reagent/first reagent/component
complexes. In a preferred embodiment, first reagent is a murine monoclonal
antibody, and second reagent is a biotinylated anti-mouse immunoglobulin.
Unreacted biotinylated second reagent may then be removed. The suspension
containing the biotinylated second reagent/first reagent/component
complexes is next reacted with insolubilized avidin in order to associate
or bind the avidin to the biotin and thereby form insolubilized
avidin/biotinylated second reagent/first reagent/component complexes. The
insolubilized avidin can take the form of an avidin-coated gel that is
packed inside a chromatography column. Then the remainder of the
suspension is separated from the insolubilized avidin complex, thereby
removing the targeted component by virtue of its incorporation into
insolubilized avidin/biotinylated second reagent/first reagent/component
complexes. The resulting suspension can be employed in bone marrow and
other transplants.
The solid support for the insolubilized avidin can take various forms,
including fibers, mesh, or tubing, and can be housed in other flow-through
devices such as extracorporeal cartridges in systems for continuously
removing selected components from a patient's bloodstream. Solid supports
for the insolubilized avidin can also take the form of magnetic beads such
as those disclosed in Reynolds, C.P., et. al., Transplantation Proceedings
XVII(1):434-436, February 1985, hereby incorporated by reference.
An improved immunoselection technique is provided by dissociating and
recovering the targeted component from the insolubilized avidin. Adherent
target cells can be conveniently dissociated by agitating the insoluble
matrix to which they are bound. In other embodiments the first and second
reagents in the separated insolubilized avidin/biotinylated second
reagent/first reagent component complexes are selectively dissociated
using available chemical techniques. For example, dithiothreitol (DTT) can
be employed to dissociate disulfide bridges such as occur in large IgM
antibodies and thereby effectively dissolve either or both of the antibody
reagents to release the target cells from the column while nonspecifically
bound adherent cells remain on the gel. Alternatively, linkages that are
specifically cleavable by enzymatic or chemical agents can be inserted
between the biotin molecule and the second reagent. Examples of such
linkages include peptide bonds cleavable by various peptidases,
disaccharide linkages cleavable by disaccharidases, or chemical bonds that
can be selectively broken under mild reducing, oxidizing, acidic, or basic
conditions.
Also provided are clinical kits for removing a specific cell population
from a heterogeneous cell suspension. The kits typically contain
insolubilized avidin in a presterilized column or cartridge housing, one
or more first reagents directed to the specific cell population, and a
biotinylated second reagent directed against the first reagents(s).
Clinical kits providing avidin bound to magnetic beads are also provided
for practicing the invention.
FIG. 8 illustrates a system 20 for treating a patient 22 so as to remove
specific cell populations such as immunoregulatory cells or tumor cells
from the patient's circulating system 32 via a double-lumen central venus
catheter 34. Of course, other known means of blood access, such as
arteriovenous shunts, fistulas, or double venipuncture, may be similarly
employed. Catheter 34 includes inlet conduit 36 and outlet conduit 38
whose respective ends 40 and 42 are adapted for insertion into the
circulatory system 32 of the human or animal patient 22. Outlet conduit 38
of catheter 34 is coupled by connector 44 to tube 46 such that whole blood
from patient 22 flows from outlet conduit 38, through tube 46, and into
cell centrifuge 24.
Cell centrifuge 24 may be any suitable device for separating the plasma and
cellular components of whole blood such that the nucleated cell fraction
of blood, the so-called buffy coat layer, that is enriched with white
blood cells of all types, generally termed leukocytes herein, can be
isolated and delivered into tube 30. The remaining blood constituents,
including substantially all of the erythrocytes (red blood cells, RBC) and
platelets, and most of the plasma, flow out of the cell centrifuge 24 into
tube 48. A suitable anticoagulant, such as heparin, is provided to the
cell centrifuge through inlet 50 to prevent clotting.
Cell centrifuge 24 may process a continuous flow of blood received from
tube 46 and deliver continuous flows of heterogeneous leukocytes and other
blood constituents into tubes 30 and 48, respectively. Alternatively, an
intermittent flow centrifuge 24 may be employed to collect the buffer coat
layer from one unit or other preselected volume of the patient's blood at
a time. Of course, the disclosed system 20 is also suitable for off-line
use, as establishing direct fluid communication between the patient's
blood at a time. Of centrifuge 24 is not a prerequisite for practicing the
invention. Thus, the selection of an appropriate treatment protocol can be
prescribed by the attending physician on the basis of the patient's
physical condition and the equipment and other facilities available at the
hospital or laboratory.
As described in detail below, the buffy coat fraction flows through tube 30
into avidinized column 28. As the leukocyte-containing fraction transits
along tube 30, one or more reagents from one or more reagent reservoirs 26
and 52 are introduced into tube 30. Mixing and incubation of the
introduced reagent(s) with the leukocytes can be facilitated by providing
one or more incubation reservoirs 54 and 56 in tube 30. After further
processing in avidinized column 28, the treated leukocyte fraction flows
through tube 58 into drip chamber 60, which also receives, from tube 48,
the plasma containing erythrocytes and platelets. The fluids from tubes 58
and 48 are allowed to mix in drip chamber 60, and the thus reconstituted
blood flows out of chamber 60 through tube 62. Tube 62 is joined, in this
on-line embodiment, by connector 64 to inlet conduit 36 of catheter 34. In
this way the reconstituted blood in tube 62 flows back to the patient's
bloodstream 32. In a typical treatment session, the equivalent of one
volume of the patient's bloodstream 32 is passed through system 20.
Treatment of the patient's blood, to selectively remove a specific
component subpopulation of the heterogeneous leukocyte population, occurs
as the buffy coat fraction passes through tube 30 and column 28. In one
representative embodiment, system 20 is provided with first and second
reagent reservoirs 26 and 52. First reservoir 26, which is disposed
upstream from both second reservoir 52 and column 28, dispenses into tube
30 a predetermined amount of a nonbiotinylated first reagent having
binding specificity for the targeted blood component. Downstream from
first reagent reservoir 26, an incubation reservoir 54 may be provided in
conduit 30 to temporarily pool the buffy coat fraction and the first
reagent in order to promote the formation of first reagent/component
complexes. The fluid in conduit 30 is thereafter reacted with a
biotinylated second reagent, having binding affinity for the first
reagent, that is introduced in metered dosage from the second reagent
reservoir 52. Downstream, a second incubation reservoir 56 may similarly
be provided in conduit 30 in order to promote the formation of second
reagent/first reagent/component complexes. Thereafter, the thus-far
processed fluid is directed through column 28, which contains
insolubilized avidin that selectively binds biotin and thereby removes
biotinylated second reagent/first reagent/component complexes from the
buffy coat fluid.
In other representative embodiments, only a single reagent reservoir 26
need be employed. For example, reagent reservoir 26 can deliver a mixture
of the first reagent and the biotinylated second reagent into tube 30.
Alternatively, reagent reservoir 26 can deliver a biotinylated first
reagent into tube 30, in order to form biotinylated first
reagent/component complexes that will selectively bind to the avidinized
surfaces in column 28.
Since any free biotinylated second (or first) reagent can also bind to the
avidinized sites in column 28, the efficiency of column 28 can be enhanced
by promoting complete association of the biotinylated reagent with the
targeted component during the transit through conduit 30. Thus, the avidin
sites in column 28 will be efficiently used to productively remove only
biotinylated component complexes from the column. One way of approaching
this objective is to provide one or more incubation chambers 54 and 56 in
conduit 30, as discussed above. Also, inflow of the first and second
reagents from reservoirs 26 and 52 can be metered in order to insure that
one or both of the reagent concentrations are below the saturation
concentration of the targeted leukocyte subpopulation in the patient's
buffy coat fraction. In addition, the downstream incubation reservoir 56
can take the form of a dialyzer device having a membrane with pores sized
to permit and promote passage of free biotinylated antibody, but not
cellular constituents, out of conduit 30 for disposal.
Column 28, as discussed above, can take the form of a sterilized
chromatography column or cartridge or cartridge housing containing an
inert support to which avidin has been insolubilized. The inert support
may be silica, glass, plastic, or any other nonreactive or nondegradable
material, and may be in the shape of beads or in other suitable shapes.
Fluid flow through column 28 may be ascending, as shown here, or
descending, and may be driven by a pumping mechanism, which may be
incorporated into centrifuge 24, or by gravity flow, particularly for
off-line applications.
FIG. 9 shows an embodiment of avidinized column 28' that is based upon
available hollow fiber technology. The cartridge housing 66, made of an
inert material such as plastic, is configured so that a fluid flow path 68
can be established between tubes 30 and 58 in system 20. At least a
portion of flow path 68 is made up of semipermeable tubing 70 that, like a
dialysis membrane, is selectively porous such that antibodies, but not
cells, can diffuse through tubing 70 into a surrounding reservoir 72. For
use in the subject immunoselection process, reservoir 72 contains water or
preferably a saline solution at a concentration selected to simultaneously
promote the selective diffusion of free biotinylated reagent and yet avoid
osmotic shock to the cells that remain in flow path 68'. Reservoir 72 may
be closed or, as shown here, provided with an inflow port 74 and an
outflow port 76, so that the dialysate or other fluid in reservoir 72 can
be continuously or periodically replenished. The semipermeable hollow
fibers 70 are typically disposed in parallel array within cartridge 28' so
as to maximize contact of the biotinylated buffy coat fraction from tube
30 with the semipermeable membrane 70 that is in intimate contact with the
dialysate in reservoir 72. The provision of avidinized surfaces in
cartridge 28 can follow several strategies, depending upon usage. In one
embodiment, avidin is insolubilized on the inner walls of dialysis tubing
70, that is, on the porous surfaces in contact with fluid flow path 68'.
In another embodiment, avidin-coated surfaces, such as beads, fibrous
meshworks, or the like (not shown), are provided in the flow path 68"
downstream from the semipermeable membranes 70. The latter embodiment is
considered preferred for the purpose of removing free biotinylated reagent
prior to contacting the cellular fraction with the insolubilized avidin.
If avidin-coated beads are housed within cartridge 28', a stainless steel
screen 78, sized to permit passage of cells, but not the beads, can be
provided in the flow path 68 downstream from wherever the beads are
sequestered.
In another embodiment, cartridge 28' is not provided with any avidinized
surfaces and is employed simply as a dialyzer to remove free biotinylated
reagent prior to contacting the buffy coat fraction with an avidinized
column 20. As such, this embodiment of cartridge 28' could replace
incubation reservoir 56 in system 20. In order to substantially inhibit
back diffusion of free biotinylated reagent from reservoir 72 into flow
path 68', the outside surfaces of the dialysis tubing 70 may be
avidinized. Alternatively, reservoir 72 may be packed with avidin-coated
beads. It is further contemplated that the two foregoing embodiments of
cartridge 28' can be coupled in series in system 20, with the one
containing avidinized surfaces in flow path 68 being disposed downstream
from the other, which serves only as a dialyzer. For convenience, the
foregoing dialyzing and cell collecting embodiments can also be
manufactured within a single housing 66.
In the subject immunoselection process, a cartridge 28' containing
avidinized surfaces in flow path 68 can be used to selectively remove
biotinylated component complexes from the heterogeneous leukocyte fraction
of mammalian blood. For example, tumor cells can be thereby selectively
removed from the bloodstream of patients with abnormally high leukocyte
titers, as in certain leukemias, after which treatment cartridge 28'
containing bound tumor cells will typically be immediately disposed.
Cartridge 28' can also be employed for positive immunoselection. For
example, after the targeted leukocyte subpopulation becomes bound, by
virtue of the disclosed biotinylated reagent(s), to the avidinized
surfaces in flow path 68, cartridge 28' is removed from system 20' and the
bound cells recovered. Physiological saline solution is first run through
flow path 68, to rinse any unbound material from cartridge 28'. Then the
flow rate of the rinsing solution can be increased, by some ten to twenty
times, in order to dissociate the targeted cells from the insolubilized
avidin.
In many cases, expansion of such positively selected cell populations will
be desirable prior to reinfusion into the patient. Cartridge 28' is
eminently suited to serve as a bioreactor for this purpose as well. For
service as a bioreactor, the avidinized surfaces should be located within
the semipermeable fibers 70, within flow paths 68'. After serving to
selectively remove the targeted cell population, e.g., immature
lymphocytes, from the patient's blood, cartridge 28' is removed from
system 20 in order to expand the population of harvested cells in situ.
For this purpose, a nutrient fluid containing a growth factor, such as an
interleukin, is circulated through flow path 68 and/or reservoir 72 in
order to promote mitosis, differentiation or activation of the harvested
cells into what in many cases will be more mature cells, e.g., cytotoxic
killer cells, that can be used for therapeutic purposes. After suitable
expansion, the bound cells and any progeny cells are flushed from
cartridge 28' for reinfusion into the patient. As an auxiliary step in any
of the disclosed positive selection strategies, the harvested cells can be
subjected to one or more additional immunoselection passages, using the
same or another first reagent, in order to assure the homogeneity of the
selected cells.
Such positively selected cells can serve many therapeutic purposes.
Immature populations of peripheral lymphocytes can be collected for in
vitro exposure to growth factors suitable to direct their maturation and
expand their population prior to autologous reinfusion into the patient.
For example, lymphocyte activated killer cells (LAK) can be raised in this
manner for cancer therapy. Killer cells have also been suggested for AIDS
therapies. Other components of cellular immunity can be similarly isolated
and enlisted, for example, certain suppressor lymphocytes for treating
thyroid diseases, T4 lymphocytes for treating AIDS, certain subpopulations
of lymphocytes for multiple sclerosis, and certain macrophages for
attaching the chloresterol plaques associated with atherosclerosis. From
bone marrow, hematopoietic stem cells can be selectively recovered for
treating cancer, hematological disorders, or genetic diseases by gene
therapy. For example, once collected in cartridge 28', the stem cells can
be exposed in situ to off-line percolation of viral or other vectors for
gene transfer and subsequent selection of transfected cells.
Abnormalities in lymphocyte populations involving excessive numbers of
circulating cells can be treated by negative cell selection using the
subject method and equipment, either on-line or off-line. Illustrative
examples include the possibility of treating rheumatoid arthritis by
reducing the circulating titer of T-helper cells. Similarly, circulating
viruses and parasites can be removed from blood using the subject method.
Tumor cells and T cells can be removed from bone marrow.
The subject method can also be also to enhance the efficiency of monoclonal
antibody production techniques. For example, peripheral blood lymphocytes
can be reacted with a first reagent in the form of an anticlonotypic
antibody that mimics a tumor associated antigen, in order to isolate
lymphocytes reactive to the tumor cells. The isolated lymphocyte
subpopulation can be fused with transformed cells to produce hybridomas.
Since the B cells are preselected using the subject method, the massive
screenings of fused cells that are typically required to select
appropriate hybridomas can be eliminated, with attendant potential for
more directly and expeditiously customizing antibodies for particular
patients. Similarly, the first reagent can be antigen, to isolate
lymphocytes reactive with the antigen, in which case the second reagent
can be a biotinylated antibody directed against a separate epitope on the
first reagent.
In a similar manner, the first reagent can be a sterilized viral particle,
in order to harvest cells specifically reactive with the virus for in
vitro expansion in the presence of the antigen and interleukin-2.
The subject method can also be employed as a sensitive assay for detecting
cancer remission or metastases, by monitoring for the presence of tumor
cells in a patient's bloodstream or bone marrow.
Another direct application is to remove cells that may be implicated in
causing or inducing tissue rejection from blood and blood constituents
prior to nonautologous transfusion. For example, Class II (Ia) bearing
cells can thereby be eliminated from platelet concentrations, in order to
reduce or eliminate the immunogenicity of the platelets, prior to
tranfusion, or to provide allogeneic tissue suitable for inducing a state
of tolerance in the recipient prior to the transfusion or transplantation
of other tissues or organs. After incubation with the first and second
reagents, e.g., mouse anti-Ia antibody and biotinylated antimouse
antibody, the whole blood or platelet concentration may be passed through
avidinized column 28, or simply transferred to a plastic storage bag
having avidinized inner walls, for complete and selective removal of the
targeted cells.
The following Examples are presented to illustrate the advantages of the
present invention and to assist one of ordinary skill in making and using
the same. The following Examples are not intended in any way to otherwise
limit the scope of the disclosure or the protection granted by Letters
Patent hereon.
EXAMPLE 1
Biotinylation of antibody 2H7
A purified murine monoclonal IgG2a antibody (2H7) to the human B cell
differentiation antigen, Bp32(CD20), was biotinylated as follows. Antibody
2H7 (Genetic Systems Corporation, Seattle, Wash.) was dialyzed in 0.1 M
NaHCO.sub.3 and adjusted to a concentration of 1 mg/ml.
N-Hydroxysuccinimidobiotin (Sigma Chemical Co., St. Louis, Mo.) was
dissolved in dimethylsulfoxide (DMSO) at a concentration of 1 mg/ml.
Unless otherwise specified below, 60 ul of this biotin solution were added
to each ml of the antibody solution and incubated for 4 hours at room
temperature. The mixture was then dialyzed in phosphate-buffered saline
(PBS; 8 mM Na.sub.2 HPO.sub.4, 0.15 mM KH.sub.2 PO.sub.4, 0.137 M NaCl,
2.7 mM KCl, pH =7.4) and stored at 4.degree. C.
EXAMPLE 2
AVIDIN CONJUGATION TO SLID PHASE SUBSTRATE
Cyanogen bromide activated Sepharose 6 MB (Pharmacia, Piscataway, N.J.) was
swollen for 35-45 min in 1 mM HCl. The gel was washed with additional 1 mM
HCl, followed by 0.1 M NaHCO.sub.3 in 0.5 M NaCl at pH =8.3 ("coupling
buffer"). Avidin Sigma Chemical Co.) was dissolved and dialyzed in
coupling buffer at a concentration of approximately 1 mg/ml and then
continuously mixed with the gel for 1 hour at room temperature. The eluate
fraction was collected, and the efficiency of conjugation (which always
exceeded 95%) was determined by measurement of protein concentration. The
concentration of avidin linked to the gel varied between 50 ug/ml and 2
mg/ml of gel. After washing the gel in an alternating fashion with 0.1 M
sodium acetate in 0.5 M NaCl at pH =4.5 ("washing buffer") and coupling
buffer, 0.2 M glycine in coupling buffer was added to block unconjugated
sites. After washing, the gel was stored in PBS with 0.1% sodium azide at
4.degree. C.
EXAMPLE 3
PREPARATION AND USE OF AVIDIN-INSOLUBILIZED COLUMN
The avidin-conjugated gel from Example 2 was packed in Pharmacia K9/15
columns (Pharmacia). Fifteen million treated cells (see below) were
suspended in a solution (PBS/BSA) of 0.5 ml PBS with 2% bovine serum
albumin (BSA). The cell suspension was continuously passed through a 3 ml
column bed of the avidin-conjugated gel at a flow rate of approximately
0.5 ml/min using PBS/BSA until a total of 10 ml of eluate was collected.
By trypan blue dye exclusion, cell viability was judged to be greater than
98%. To regenerate the column, the gel was extensively washed with PBS
(without BSA) and mechanically agitated using a Pasteur pipette to
dislodge adherent cells. The gel was then sequentially washed three times
with washing buffer and coupling buffer and stored in PBS with 0.1% sodium
azide at 4.degree. C.
For certain experiments the K9/15 column including rubber stopper and
tubing was sterilized with ethylene oxide. The column was then packed with
the avidin-conjugated gel and allowed to incubate for 30 min with 0.05%
formaldehyde (V/V) in PBS. The formaldehyde was then removed by extensive
washing with PBS. No loss of immunoadsorption efficiency occurred using
this method of sterilization.
EXAMPLE 4
Intracellular Labeling of Tumor Cells
Daudi is a human lymphoblastoid B cell line originally derived from a
patient with Burkitt's lymphoma. Cancer Res. 28:1300, 1968. The Daudi cell
line was maintained in our laboratory by serial passage in tissue culture
medium containing RPMI 1640 with 10% fetal calf serum, sodium pyruvate,
L-glutamine, streptomycin, and penicillin. Indirect immunofluorescence
staining and analysis (as described in J.Exp.Med. 110:875, 1959) using a
fluorescence-activated cell sorter (FACS IV; Becton Dickinson, Mountain
View, Calif.) confirmed that 100% of the Daudi cells bear surface IgM.
These studies also demonstrated that all cells expressed the pan-B cell
marker Bp32(CD20).
Intracellular labeling of Daudi cells with fluorescein isothiocyanate
(FITC) was performed using a modification of the procedure described in J.
Immunol. Methods 37:97, 1980. Briefly, Daudi cells were incubated with 60
ug/ml FITC (Sigma Chemical Co.) in PBS for 20 min at 37.degree. C. Cold
PBS was added to stop labeling, and the cells were centrifuged through an
underlayer of fetal calf serum to remove excess FITC. Cells were then
washed twice with PBS. This method produced bright intracellular
fluorescence in 100% of the cells. Furthermore, cell viability assessed by
trypan blue exclusion was identical in FITC-labeled Daudi cells as
compared to unlabeled Daudi cells. Immunofluorescence staining with
rhodamine isothiocyanate-conjugated antibody 2H7 demonstrated that cell
surface expression of Bp32(CD20) was unaffected by the FITC-labeling of
Daudi cells.
EXAMPLE 5
Treatment of Cells with Biotinylated Antibodies
In addition to the antibody 2H7 described in Example 1, the following
antibodies were used: A purified murine monoclonal IgG2a antibody
(DM1;Becton-Dickinson, Mountain View, Calif.) specifically recognizes the
u heavy chain of human immunoglobulin. A monoclonal IgG2a antibody (9E8;
Genetic Systems) recognizes the p(15)E antigen of murine leukemia virus
and is not reactive with human tissues including bone marrow or Daudi
tumor cells. Affinity purified goat antimouse immunoglobulin (GAMIg),
biotinylated goat antimouse immunoglobulin (B-GAMIg), and biotinylated
F(ab').sub.2 goat antimouse immunoglobulin (B-F(ab').sub.2 -GAMIg) were
obtained from Tago, Inc., Burlingame Calif.
Bone marrow mononuclear cells were obtained by Ficoll-Hypaque (specific
gravity =1.077; Pharmacia) density gradient centrifugation from bone
marrow samples (5-10 ml) obtained from healthy donors.
Concentrations of antibodies utilized in these experiments were based on
immunofluorescence staining and FACS analysis which demonstrated that a
minimum of 50-100 ug/ml of unconjugated or biotinylated antibody 2H7 or
5-10 ug/ml of antibody DM1 1 were respectively required to saturate the
binding sites for Bp32 or u on Daudi cells (data not shown).
BIOTINYLATED ANTIBODY 2H7
Fifteen million cells per ml of bone marrow contaminated with 5-10% Daudi
cells were incubated for 30 minutes with biotinylated antibody 2H7 in
PBS/BSA at 4.degree. C. Cells were then washed twice with PBS/BSA before
column treatment.
Biotinylated Goat Antimouse Antisera
Fifteen million cells per ml of a similar bone marrow and Daudi cell
mixture were incubated for 30 minutes with either antibody 2H7 or antibody
DM1 is PBS/BSA at 4.degree. C. Cells were washed twice after incubation
with monoclonal antibody before and twice again after incubation with
B-GAMIg or B-F(ab').sub.2 -GAMIg for 30 minutes at 4.degree. C.
EXAMPLE 6
Detection of Residual Tumor Cells
A known number of the bone marrow mononuclear cells admixed with a known
number of the labeled Daudi cells from Example 4 were placed into 96 well
flat-bottom microdilution plates (Costar, Cambridge, Mass.) and examined
with a Leitz inverted fluorescence microscope. A series of preliminary
experiments demonstrated that a single FITC-labeled Daudi cell could be
detected in a well containing a total of one million cells, representing a
tumor cell contamination of 0.0001%.
Unless otherwise specified below, aliquot samples of 1000 pretreated cells
were placed in microdilution wells; after column treatment, 100,000
treated cells were placed in neighboring wells for comparison. The numbers
of FITC-stained cells were counted in three pretreated and three treated
wells and averaged to determine the number of tumor cells per 1000 total
cells before column treatment and the number of tumor cells per 100,000
cells in the treated group, respectively. Total cell counts were performed
with a hemacytometer. The Log Removal of Daudi cells was calculated from
absolute cell counts: The relative percentage of FITC-labeled cells before
and after column treatment, indicating the removal of tumor cells, was
determined as follows:
##EQU1##
Percentage of Recovery was determined as follows:
##EQU2##
For example, the following calculations are from an experiment treating a
mixture of bone marrow and FITC-labeled Daudi cells successively with 10
ug/ml antibody DM1 and 1:100 dilution of B-GAMIg, and then passing the
treated cells over a 3 ml column bed of Sepharose containing 1 mg/ml of
avidin. The pretreatment sample had a mean of 58 FITC-labeled cells (52,
59, 62 labeled cells counted in 3 separate pretreatment wells) per 1,000
cells (or 5.8% FITC-labeled cells); while the posttreatment sample
contained a mean of 21 FITC-labeled cells (19, 22, 22 labeled cells
counted in 3 separate wells) per 10.sup.5 cells (or 0.021% FITC-labeled
cells). There were a total of 15.0.times.10.sup.6 cells in the
pretreatment sample, and 11.4.times.10.sup.6 cells in the post treatment
sample.
##EQU3##
EXAMPLE 7
Assay System for Hematopoietic Progenitors
The assay system for culturing committed granuloctye-monocyte progenitors
(CFU-C) described in Exp. Hematol. 6:114, 1978, was followed. Briefly,
1.0.times.10.sup.5 cells were cultured in triplicate in 1 ml aliquot
mixtures containing conditioned medium from phytohaemagglutinin-(Wellcome
Labs, Oxford, England) stimulated peripheral blood lymphocytes, agar
(Bactoagon, Difco, Detroit, Mich.), and MEM alpha medium (Gibco, Grand
Island, N.Y.). Colonies were counted after 14 days incubation at
37.degree. C. in 5% CO.sub.2 using an inverted microscope.
EXAMPLE 8
Depletion of Daudi Cells with Biotinylated Antibody 2H7
FIG. 2 summarizes the results from a series of experiments in which fifteen
million bone marrow cells containing 5-10% Daudi cells were treated with
100 ug/ml biotinylated antibody 2H7 and then passed over a 3 ml column bed
of Sepharose 6MB linked to the indicated amount of avidin. Avidin
concentrations (ug/ml) is plotted on the abscissa. Log Removal (solid
circles) and Percentage Recovery (open circles are plotted on the
ordinates. Each data point represents the results of two to four separate
experiments.
FIG. 2 shows that increasing concentration of avidin on the gel was
correlated with improved removal of Daudi cells. Avidin concentrations of
less than or equal to 100 ug/ml resulted in less than one log depletion,
while concentrations of 1000 ug/ml were able to produce over two log
removal of Daudi cells from bone marrow. However, increasing the
concentration of avidin on the gel also caused decreased recovery of bone
marrow cells.
Attempts were made to improve the recovery of bone marrow cells by
decreasing the biotin/antibody conjugation ratio. Biotin/avidin conjugates
were prepared by incubating antibody 2H7 (1 mg/ml) with the concentrations
of N-Hydroxysuccinimidobiotin indicated in TABLE 1 for four hours. Fifteen
million bone marrow cells containing 5-10% Daudi cells were incubated for
30 minutes with 100 ug/ml biotinylated antibody 2H7 and passed over 3 ml
column bed of Sepharose containing 1 mg/ml avidin. TABLE 1 shows the
effect of varying the degree of biotinylation of the specific antibody
upon log removal of the specific targeted subpopulation and upon
percentage of recovery of the re | | |
