Release of cells from affinity matrices

The present invention comprises a process for release from the cell-receptor complex of positively selected cells in viable, functional condition, where a ligand involved in the particular receptor-ligand interaction utilized for the affinity purification is selectively attacked by one or more degradative enzymes specific for that ligand. A resulting cell suspension can be obtained substantially free of receptor material. This invention, in one embodiment, contemplates a method for positive stem cell selection, utilizing anti-MY10 and immunomagnetic microspheres to isolate CD34-positive marrow cells and employing an enzyme to release micropheres from the isolated CD34-positive cells. Reproducible enzymatic cleaving of immunomagnetic microspheres from MY10-positive cells can be achieved by brief treatment of the preparation with papain or chymopapain. The isolated CD34-positive cells are particularly desirable for bone marrow transplantation.

FIELD OF THE INVENTION 
The invention is directed toward a method of releasing viable cells from 
cell-receptor affinity complexes. 
BACKGROUND OF THE INVENTION 
Bone marrow transplantation (BMT) is now an important treatment modality 
for aplastic anemia and leukemia, and BMT strategies are under intense 
investigation for utility in other malignancies and in genetic disease. 
Two forms of bone marrow transplantation have been developed, namely, the 
allogeneic (from a genetically different donor) and autologous (using 
marrow cryopreserved prior to ablative therapy) forms. Both are based on a 
principle of high dose chemotherapy and/or radiation therapy followed by 
repopulation of the marrow by infusion. 
Due to the inability to transfer only the stem cell population, the 
applicability of allogeneic BMT remains restricted by graft vs. host 
disease (GVHD), which is apparently mediated by T lymphocytes in the graft 
cell population. Risk of GVHD has limited allogeneic BMT to use only in 
highly fatal diseases, and even then, only for patients with HLA-matched 
donors, usually siblings. Autologous BMT can avoid most of the problems 
associated with allogeneic transplants. In autologous BMT, however, it is 
necessary to reintroduce only desirable cell populations free of diseased 
cell populations (e.g., occult tumor cells) to avoid re-introduction of 
the disease. 
The problems associated with both allogeneic and autologous BMT can be 
alleviated by using purified stem cell populations for the graft. These 
purified populations can be obtained from marrow cell suspensions by 
positive selection (collecting only the desired cells) or negative 
selection (removing the undesirable cells), and the technology for 
capturing specific cells on affinity materials is well developed. (Wigzel, 
et al. (1969), J. Exp. Med., 129:23; Schlossman, et al. (1973), J. 
Immunol., 110:313; Mage, et al. (1977), J. Immunol. Meth., 15:47; Wysocki, 
et al. (1978), Proc. Nat. Acad. Sci., 75:2844; Schrempf-Decker, et al. 
(1980), J. Immunol. Meth., 32:285; Muller-Sieburg, et al. (1986), Cell, 
44:653.) 
Monoclonal antibodies against antigens peculiar to mature, differentiated 
cells have been used in a variety of "negative" selection strategies to 
remove undesired cells (i.e., to deplete T cells or malignant cells from 
allogeneic or autologous marrow grafts, respectively). (Gee, et al., 
J.N.C.I. (1988) 80:154-9; Gee, al., "Proc. of 1st Int. Workshop on Bone 
Marrow Purging," in Bone Marrow Transpl., Supp. 2, London, MacMillan, 
1987.) Successful purification of human hematopoietic cells by negative 
selection with monoclonal antibodies and immunomagnetic microspheres has 
been reported which involved use of multiple monoclonal antibodies, thus 
making it more costly for clinical application than positive selection. 
(Griffin, et al., Blood, 63:904 (1984); Kannourakis, et al., Exp. 
Hematology, 15:1103-1108 (1987).) Most studies report 1 to 2 orders of 
magnitude reduction in the target cell level following monoclonal antibody 
treatment. This may not be adequate T lymphocyte depletion necessary to 
prevent GVHD in allogeneic transplants, and it is certainly insufficient 
in autologous bone marrow transplantation where 10.sup.6 to 10.sup.9 
malignant cells may be present in the patient's marrow. 
Positive selection of normal marrow stem cells is an alternative for 
treatment of the marrow graft. The procedure employs a monoclonal antibody 
which selectively recognizes human lymphohematopoietic progenitor cells, 
such as the anti-MY10 monoclonal antibody that recognizes an epitope on 
the CD34 glycoprotein antigen. Cells expressing the CD34 antigen include 
essentially all unipotent and multipotent human hematopoietic 
colony-forming cells (including the pre-colony forming units (pre-CFU) and 
the colony forming unit-blasts (CFU-Blast)) as well as the very earliest 
stage of committed B lymphoid cells, but NOT mature B cells, T cells, NK 
cells, monocytes, granulocytes, platelets, or erythrocytes. See Civin, 
U.S. Pat. No. 4,714,680. 
CFU yields in MY10-positive cell populations are far higher than the 
0.1-23% range of recovery of CFU observed after treatment of marrow grafts 
with 4-hydroperoxycyclophosphamide, a cyclophosphamide metabolite that 
"purges" malignant cells from marrow grafts without ablating the ability 
of the marrow to engraft. (Yeager, et al. (1986), N. Eng. J. Med., 
315:141.) Positive selection utilizing CD34 monoclonal antibody also 
appears more feasible (over the long term) for BMT than negative selection 
strategies for isolations of rare progenitor cells from marrow or blood, 
offering advantages such as specificity, simplicity, and cost in treatment 
of diseases other than leukemia. 
Recently, Berenson, et al. (1986), J. Immunol. Meth., 91:11-19, disclosed a 
method for large scale positive selection of class II antigen-positive or 
CD34-positive cells from marrow, using monoclonal antibody columns. The 
preliminary results were based on in vitro CFU assays on separated human 
marrow samples, and actual in vivo BMT experiments in primates. (Berenson, 
et al. (1988), J. Clin. Invest., 81:951-960.) The primate experiments were 
possible, since some epitopes of the MY10 glycoprotein are shared between 
humans and primates. 
Marrow cells tend to aggregate nonspecifically at the high cell density 
that results from slow percolation of marrow through the column 
necessitated by the relatively low avidity of monoclonal antibody for cell 
surface antigen, so this work took advantage of the high affinity 
avidin-biotin interaction. Marrow cells were first labelled with 
monoclonal antibody, then with biotin-labelled anti-mouse Ig. Upon 
percolation through a column of avidin-coated macroscopic agarose beads, 
antigen-positive cells bound to the column, even at high flow rates. After 
washing of the column to remove unbound cells, bound cells were physically 
sheared from the beads by vigorous pipetting of the column contents. This 
release method does not guarantee that all cell-antibody complexes (i.e., 
antibody-coated cells) were eliminated from the final cell suspension. 
Further refinement of techniques for positive selection of MY10-positive 
cells are available which do not require treatment of marrow cells with 
multiple reagents (CD34 monocional antibody, biotinylated polyclonal 
anti-mouse antibody, avidin-conjugated macrobeads). Magnetic microspheres 
with low nonspecific avidity for cells are commercially available, either 
in uncoated form (for adsorption of the desired antibody) or coated with 
anti-mouse Ig. Cell trapping can more readily be avoided with monodisperse 
microspheres, and the immunomagnetic microsphere technique has been shown 
to be effective for positive selection in, e.g., Gaudernack, et al., J. 
Immunol. Meth., 90:179 (1986). 
The most desirable cell suspension for BMT would be one that is 
substantially free of cell:receptor complexes. Thus, the problem of how to 
release positively selected cells from the affinity matrix once they have 
been separated from the non-selected cells still remains. 
SUMMARY OF THE INVENTION 
It is an object of this invention to provide a method for release of cells 
bound to affinity matrices which preserves the viability and function of 
the cells. 
It is also an object of this invention to provide a method for release of 
positively selected cells from the receptors used in selecting them, so 
that the cells remain in viable, functional condition. 
It is a further object of this invention to provide a method for the 
recovery of viable, functional bone marrow cells characterized by the CD34 
surface antigen which are substantially free of all foreign protein, 
particularly antibodies to the CD34 antigen. 
These and other objects may be achieved by the practice of the invention 
disclosed herein. 
The present invention comprises a process to release positively selected 
cells in viable, functional condition, where a ligand involved in the 
particular receptor-ligand interaction utilized for the affinity 
purification is selectively attacked by one or more degradative enzymes 
specific for that ligand. 
The exact type of selective attack can be controlled by the enzyme 
selection to be non-toxic and non-injurious to the cells in question and 
directed to a limited number of cell surface structures. By attacking the 
cell surface "ligand," rather than the receptor on the affinity matrix, 
the cells are freed of the "foreign material" which had coated their 
surfaces, at the cost of only a minor nick in certain exposed membrane 
molecules. The resulting cell suspension is substantially free of receptor 
material. For some in vitro processes and procedures receptor-carrying 
cells may be suitable, but for in vivo processes and particularly for 
therapeutic purposes, these receptors can be extremely detrimental. 
This invention, in one embodiment, contemplates a method for positive "stem 
cell" selection, utilizing anti-MY10 and immunomagnetic microspheres to 
isolate CD34-positive marrow cells and employing an enzyme to release 
microspheres from the isolated CD34-positive cells. Reproducible enzymatic 
cleaving of immunomagnetic microspheres from MY10-positive cells can be 
achieved by brief treatment of the preparation with papain or chymopapain. 
The chymopapain treatment does not produce detectable damage to human 
colony-forming cells or rat stem cells. When employing CD34-positive 
monoclonal-antibody-coated microspheres, this immunomagnetic microsphere 
technique has fewer steps than the avidin-biotin system.

DETAILED DESCRIPTION OF THE INVENTION 
This invention contemplates release of cells from receptors bound 
specifically to their surfaces. The cells contemplated by this invention 
comprise animal cells, preferably mammalian cells, where the cells are 
characterized by the presence, on their surface, of surface 
ligands--molecules which comprise one or more binding sites for particular 
receptors. These ligands are peculiar to the cell-type contemplated by 
this invention and absent on other cell-types which are undesirable for a 
particular purpose. This purpose is optimally therapeutic, involving 
administration of a selected subpopulation of cells to a patient. Suitable 
cell types include stem cells from blood or bone marrow, hormone-secreting 
cells, particular types of lymphocytes, and LAK cells, as well as other 
cell types that will be apparent to those of ordinary skill in the art. 
Selection of the subpopulation comprises binding of the peculiar surface 
ligands by receptors. 
Specific cell surface ligands to which the receptors bind are 
carbohydrates, proteins, lipids, and combination molecules, including, but 
not limited to, well-known cell surface antigens, cell membrane proteins 
and the carbohydrate portion of cell surface glycolipids and 
glycoproteins. The receptors contemplated by this invention include, 
without limitation, antibodies specific for cell surface antigens, lectins 
specific for the carbohydrate portions of cell-surface glycolipids and 
glycoproteins and other proteins that bind to cell surface ligands. The 
receptors may be in their native state, they may be bound in turn to other 
binding moieties, or they may be covalently attached to another component, 
such as a fluorescent label or an insoluble support matrix. Materials that 
may be used for insoluble support matrices are well known in the art and 
include protein, carbohydrates, polystyrene, polyacrylamide, magnetic 
material, and other materials. A variety of support configurations are 
known in the art including flat surfaces, beads, microspheres and the 
like. 
Cell-receptor complexes are well-known in the art and are broadly 
contemplated for the practice of this invention. They are prepared by 
incubating the cells with the receptor in any medium that is suitable for 
maintaining cell viability, and which does not interfere with 
cell-receptor binding, for sufficient time to permit binding. These 
cell-receptor complexes are separated from other cells, which do not bind 
to the receptor molecules, by use of separation techniques based on 
properties of either the receptor molecule or the cell-receptor complex. 
Examples of the separation techniques include fluorescence-activated cell 
sorting or flow cytometry where the receptor is a fluorescent-labelled 
antibody, an avidin-affinity column where the receptor is a 
biotin-labelled antibody, and magnetic separation where the receptor is a 
murine antibody which reacts with the anti-mouse IgG on immunomagnetic 
microspheres. Separation of the cell-receptor complex is within the 
contemplation of this invention irrespective of the selection technique 
employed to obtain the complex. 
After the cell-receptor complex is separated, the cells are released from 
the complex by treatment with a degradative enzyme, where the enzyme 
specifically degrades the cell surface ligand to which the receptor is 
bound without substantially decreasing the viability or function of the 
cell population. The enzyme is selected from the group consisting of 
carbohydrases, proteases and lipases, and is usually selected based on the 
known chemistry of the cell surface ligand. Proteases may be used for cell 
surface proteins and glycoprotein antigens, and specific carbohydrases may 
be used for cell surface glycolipids and glycoproteins. Examples include a 
neuraminidase for sialic-acid-containing surface carbohydrates, 
glycosidases such as N-glycanase, O-glycanase, endo-glycosidase F, 
endo-glycosidase H and proteases such as pepsin, papain, chymopapain, 
chymotrypsin and others and phospholipases C and D. Alternatively, enzymes 
that are recognized empirically to degrade the ligand of interest or 
enzymes that have been empirically determined to release the receptor from 
cell:receptor complexes can be used. 
Effectiveness of the chosen enzyme can readily be confirmed by the 
following procedure. First, a cell population containing the cells of 
interest is incubated with the chosen enzyme (or a panel of candidate 
enzymes) under conditions that facilitate the activity of the enzyme(s) 
without compromising viability of the cells. After incubation, the cells 
are washed to remove the enzyme and tested for ability to bind the 
receptor. The cells which have been treated with the chosen enzyme are 
also tested for viability. If the enzyme treatment has destroyed receptor 
binding without reducing viability, then the enzyme is a suitable 
candidate. 
The suitability of the enzyme for the method of this invention can be 
confirmed by incubation of the cells with the receptor followed by 
treatment with the enzyme. Then the cells are washed to remove the enzyme 
and the receptor, and the cells are tested for viability. If the cells are 
released from the receptor and remain viable, then the suitability of the 
enzyme choice is confirmed. Those of ordinary skill in the art can 
routinely apply this procedure to select appropriate enzymes for release 
of cells from the many well-known and characterized cell-receptor 
complexes. 
In the practice of this invention, cell-receptor complexes, substantially 
free of unbound cells, are incubated with the selected enzyme in a medium 
suitable for suspension of the particular cell type, under gentle 
conditions of temperature and agitation selected to maximize viability of 
the cells. These conditions will be apparent to one of ordinary skill from 
the conditions used in isolation of the cells and in preparation of the 
cell:receptor complex. The incubation is continued for a period of time 
sufficient for substantial release of all receptors from the cell surface. 
The enzyme dose can be selected based on the results of routine tests 
concerning degradation of the cell-surface ligand. Increasing enzyme dose 
or increasing temperature permits reduction in the time of incubation. 
Time, temperature and enzyme dose for the incubation can be optimized to 
maximize viability of the released cells. This optimization is a routine 
procedure within the ordinary skill in the art. 
After the enzyme treatment, cells are washed to remove the enzyme and the 
receptor, and the cell population is recovered in a suitable suspending 
medium. The washing procedures used to recover the original cell 
population prior to receptor binding and separation are suitable for this 
step. The cell population, after enzyme treatment and washing, is made up 
of the positively selected cell-type and substantially free of foreign, 
antigenic material. It is therefore particularly suited for therapeutic 
use. 
In a particular embodiment of this invention, cells bearing the CD34 
antigen are positively selected to provide a population of cells for bone 
marrow transplant without any foreign receptor on their surfaces. This 
cell population contains the lymphohematopoetic progenitor cell types but 
does not contain mature cells such as mature B cells, T cells, NK cells, 
monocytes, granulocytes, platelets and erythrocytes, nor does it contain 
malignant cells. The method of this invention first requires that a 
population of CD34-positive, receptor-bound cells be obtained based on 
their ability to bind anti-MY10 antibody. Procedures to prepare such 
populations bound to the anti-MY10 antibody are taught in U.S. Pat. No. 
4,714,680 and are incorporated herein by reference. A preferred method of 
obtaining such a cell population comprises attaching the cells to 
immunomagnetic microspheres using an anti-MY10 monoclonal antibody and 
holding the microsphere-bound cells in place with a magnetic field while 
the unbound cells are washed away. 
In a representative procedure, buffy coat cells are obtained from bone 
marrow using standard techniques, with or without Ficoll purification, and 
suspended at from 5.times.10.sup.6 to 10.sup.8 cells/ml in a suitable 
tissue culture medium, such as Gibco TC199, preferably with 0.25% human 
serum albumin (HSA) present. Anti-MY10 monoclonal antibody is added at an 
amount in excess of the amount needed for labelling cells with the 
antibody, determined in separate experiments. The preferred antibody will 
form complexes with epitopes identified by the monoclonal antibody 
produced by hydridoma cell line ATCC HB-8483, identified in U.S. Pat. No. 
4,714,680. The cells are incubated with the antibody for from 10 to 120 
minutes (preferably from 20 to 30 minutes) at a temperature of from 
0.degree. to 40.degree. C. (preferably about 4.degree. C.) with gentle 
agitation. After incubation, the cells are washed one or more times by 
centrifugation using the same tissue culture medium. Then the 
antibody-treated cells are mixed with IgG-coated magnetic microspheres, 
where the IgG is specific for the species-type Ig of the anti-MY10 
monoclonal antibody used to coat the cells, usually using 0.5-4 
microspheres per cell. Incubation conditions are the same as those given 
for the incubation with anti-MY10. After the incubation, the microspheres 
and the microsphere-bound cells are held in the incubation vessel by a 
strong magnetic field and washed to remove the unbound cells. A suitable 
washing protocol would be three times with 10 volumes of tissue culture 
medium. 
An alternative, less preferred method for obtaining the desired population 
of cells bound to magnetic microspheres involves preparing magnetic 
microspheres coated with the monoclonal anti-MY10 antibody and incubating 
the original marrow isolate directly with these microspheres under the 
incubation conditions given above. This alternative process involves fewer 
processing steps, but it may yield lower recoveries of colony-forming 
cells. 
The desired cells are detached from the magnetic microspheres by treatment 
with an enzyme, preferably chymopapain. Any suitable preparation of 
chymopapain may be used. Therapeutic preparations designed for use in 
treating lumbar disc disease are particularly suitable. The cell-bound 
microspheres are treated with from 50 to 500 units of chymopapain per 
10.sup.7 cells. (1 unit hydrolyzes 1 picomole of p-nitroaniline from 
benzoylarginine-p-nitro-anilide per second.) The treatment is performed in 
a suitable tissue culture medium, preferably TC199, for from 5 to 240 
minutes, preferably 5 to 45 minutes, at from 4.degree. to 40.degree. C., 
preferably 30.degree.-37.degree. C., at a cell concentration of from 
5.times.10.sup.6 to 10.sup.8 per ml. After the incubation, the magnetic 
microspheres may be separated based on density or trapped by a magnetic 
field and the cells decanted. Preferably, the cell population is then 
freed of residual enzyme by centrifugal washing with tissue culture 
medium. The resultant cell population is particularly useful for bone 
marrow transplants. 
The cell population of this invention can be used in therapeutic methods, 
such as stem cell transplantation, as well as other therapeutic methods 
that are readily apparent to those of skill in the art. For example, such 
cell populations can be administered directly by I.V. to a mammalian 
patient requiring a bone marrow transplant in an amount sufficient to 
reconstitute the patient's hematopoietic and immune system. Precise, 
effective quantities can be readily determined by those skilled in the art 
and will depend, of course, upon the exact condition being treated by the 
therapy. In many applications, however, an amount containing approximately 
the same number of stem cells found in one-half to one liter of aspirated 
marrow should be adequate. 
The following examples are provided to illustrate specific embodiments of 
the present invention. The examples are included for illustrative purposes 
only and are not intended to limit the scope of the present invention. 
EXAMPLE 1 
Effects of Proteolytic Enzyme Treatment On Antigen Expression of KG1a Cells 
In order to determine whether chymopapain cleaved epitopes from cells, the 
effect of chymopapain was tested on the KG1a human leukemia cell line. 
KG1a cells, coated with anti-MY10 monoclonal antibody and also uncoated, 
were treated with chymopapain (200 units/ml TC199, 37.degree. C., 10 
minutes), washed, and then stained with monoclonal antibodies. Chymopapain 
treatment resulted in the almost complete removal of anti-MY10 antibody 
and the MY10 epitope from KG1a cells. Other cell surface antigens, the 
transferrin receptor and CD45R epitopes, treated as controls, were still 
detectable, through decreased, on the KG1a cells. 
EXAMPLE 2 
Effects of Proteolytic Enzyme Treatments On Colony-Forming Capacity of 
Cells from Bone Marrow 
Effective chymopapain treatment did not appear toxic to human hematopoietic 
colony-forming cells. Buffy coat preparations of marrow mononuclear cells 
(MMC) were washed with TC199 (without additives), then treated with 200 
units/ml chymopapain (or control medium) for 10-30 minutes at 37.degree. 
C. (10.sup.7 nucleated cells/ml TC199). Neither the initial viable cell 
counts nor the colony-forming capacities of the chymopapain-treated marrow 
cells were significantly different from those of controls (Table 1). 
TABLE 1 
______________________________________ 
Effect of Chymopapain Treatment 
on Colony Forming Capacity of MMC 
10.sup.7 buffy coat marrow cells were incubated for 10 or 30 min- 
utes in 1 ml TC199 culture medium containing 200 units/ml 
chymopapain, pelleted, then resuspended in an identical volume 
for colony-forming assays. 
Duration of Colonies per 10.sup.5 Cell Plated 
Chymopapain Treatment (min) 
CFC-GM BFU-E 
______________________________________ 
None 131 105 
10 114 114 
30 109 109 
______________________________________ 
In contrast, when MMC were treated with papain (conditions: 0.026 mg/ml 
papain, incubation for 90 minutes), 10% of the initial functional 
colony-forming cells were recovered. Trypsin was found to be toxic to 
hematopoietic colony-forming cells. 
EXAMPLE 3 
Binding of Cells to Immunomagnetic Microspheres Via Anti-MY10 Monoclonal 
Antibody 
The "indirect" method of incubating cells first with anti-MY10, then with 
sheep anti-mouse IgG.sub.1 -coated magnetic microspheres, was utilized in 
the current experiment. 10.sup.6 KG1a cells or MMC (10.sup.7 /ml in TC199 
tissue culture medium containing 1% human serum albumin [HSA] and 20 mg/1 
gentamycin) were incubated at 4.degree. C. for 30 minutes, on a 
hemocytology rocker-rotator mixer, with undiluted MY10 hybridoma 
supernatant (an amount previously determined to provide a condition of 
antibody excess for labelling). The cells were then washed twice by 
centrifugation (250.times.g, 10 min) with ice-cold TC199 containing 
gentamycin and 0.25% HSA. 
The monoclonal antibody-treated KG1a cells or MMC (in 1 ml TC199 containing 
gentamycin and 1% HSA) were mixed with anti-mouse IgG.sub.1 -coated 
(Dynabeads M-450 from Dynal Corp.) magnetic microspheres in a screw-top 
tube, usually at a ratio of 0.5-4 microspheres per KG1a cell or per MMC. 
The microsphere-cell mixtures were vortexed gently and incubated at 
4.degree. C. for 30 minutes, on a hematology rocker-rotator mixer. After 
incubation, the cells were separated, using a strong magnet to hold the 
microspheres and microphere-bound cells to the wall of the tube while 
unbound cells were poured from the tube. The microsphere/cell complexes 
were washed in this fashion 3 times with 10 ml TC199 without additives. 
The MY10-positive and MY10-negative marrow cell fractions were examined, 
using a phase contrast microscope, for the presence of rosettes and free 
cells, and saved for analyses as described below. 
Using Ficoll-Hypaque purified low density MMC cells, a microsphere per MMC 
ratio of 0.5:1 was found to deplete 90% of the hematopoietic 
colony-forming cells from the CD34-negative cell fraction. 
EXAMPLE 4 
Release of Microspheres From KG1a Cells Using Proteolytic Enzymes 
A series of experiments was performed, attempting to detach immunospheres 
from KG1a cells, using papain treatment for 1-3 hours over a wide range of 
papain concentrations. Treatment under these conditions with papain 
(concentration 0.026 mg/ml) resulted in essentially 100% release of 
microspheres from KG1a cells with 84-93% viable cell recovery. In 
contrast, dyspase (Boehringer Mannheim) was not effective at releasing 
immunomagnetic microspheres linked to KG1a cells by anti-MY10. Trypsin was 
not tested for efficacy at releasing microspheres from KG1a cells because 
of its toxicity to bone marrow colony-forming cells. 
As an alternative to papain, MY10/microsphere/cell complexes were treated 
with chymopapain (Chymodiactin.RTM., Flint Laboratories/Boots Co. [U.S.A], 
Lincolnshire, ILL.). These experiments showed chymopapain to be effective 
at releasing immunomagnetic microspheres from KG1a cells over a wide range 
of concentrations and times of incubation. In five experiments, treatment 
of KG1a cells (bound via anti-MY10 to immunomagnetic microspheres) using 
200 units/ml chymopapain for 10 minutes at 37.degree. C. resulted in 
90-109% (mean=100%) recovery of viable KG1a cells; treatment for 45 
minutes was only marginally more toxic. 
EXAMPLE 5 
Isolation of MY10-Positive Cells From Marrow 
MY10-positive cells were isolated using anti-MY10 and immunomagnetic 
microspheres according to Example 3 (0.5 microspheres per nucleated cell), 
and separation of MY10-positive cells from microspheres with chympapapin 
according to Example 4 (200 units/ml, 37.degree. C., 10 minutes). The 
resulting cell population was usually 50 to 90% blasts compared to 1 to 3% 
blasts in the starting cell preparation. The main contaminating cell-type 
was nucleated erythrocytes. Lymphoctyes and granulocytes were usually 
present in small numbers. MY10-positive marrow cells have been previously 
shown to have blast and early lymphoid morphology. 
EXAMPLE 6 
Light Scattering Characteristics of Isolated MY10-positive Marrow Cells 
On flow cytometry CD34-positive, lymphohematopoietic cells have light 
scattering properties characteristic of the "BLAST" and "LYMPH" windows. 
Consistent with this, the immunomagnetic microsphere enriched 
MY10-positive cell population of Example 5 contained predominantly cells 
with "BLAST" and "LYMPH" light scattering properties. The percentage of 
cells which was included in the "BLAST" window was particularly 
informative, since fewer than 10% of unseparated bone marrow cells show 
this type of light scatter, while the MY10-positive cell fraction usually 
had 60-70% of cells in the "BLAST" window. 
EXAMPLE 7 
Cell Surface Antigens of Isolated MY10-positive Cells 
Freshly islolated cell fractions of Example 5 were tested for expression of 
MY10 and other cell membrane antigens. The MY10-negative cell fractions 
were depleted (by approximately 90%) of cells expressing detectable MY10, 
as compared to unseparated marrow, indicating efficient binding of 
anti-MY10 coated cells by the microsphere procedure. The majority of cells 
in the "MY10-positive" cell fractions did not, after re-exposure to 
anti-MY10, bind the anti-MY10 monoclonal antibody (by indirect 
immunofluorescence). 
Of particular importance, other epitopes of the CD34 glycoprotein were 
found to be resistant to the chymopapain treatment. Direct enumeration of 
the CD34-positive cells in the selected cell population was thus possible 
by using monoclonal antibodies directed against these 
chymopapain-resistant CD34 epitopes. Other cell surface antigens were 
found to be still detectable after the chymopapain treatment; these 
include HLA-DR, CD3, CD4, CD5, CD14, CD19, CD20 and CD45. The retention of 
the CD3, CD4 and CD5 epitopes permits monitoring of residual T cells in 
the MY10-positive cell fraction. 
EXAMPLE 8 
Hematopoietic Colony-Forming Capacity of Isolated MY10-positive Marrow 
Cells 
The MY10-positive cell fractions of Example 5, obtained using the 
immunomagnetic microspheres, were enriched in CFC-GM (23-41-fold) and 
BFU-E (21-31-fold), with 11-45% recovery of these colony-forming cells in 
the MY10-positive cell fraction. The MY10-negative cell fractions were 
correspondingly depleted of colony-forming cells. 
To ensure that the isolation procedure did not diminish the colony-forming 
capacity of the recovered cells, the isolation procedure was run in 
parallel with another selection procedure (panning). The nearly identical 
results achieved by the two procedures (Table 2) indicate that excellent 
colony-forming capacity is retained by the MY10-positive cells following 
incubation with the microspheres and treatment with chymopapain. 
TABLE 2 
__________________________________________________________________________ 
Comparison of Immune Adherence Progenitor 
Cell Purification from Normal Human Bone Marrow 
Cells Using Immunomagnetic Beads versus "Panning" 
Panning Microspheres 
Control 
MY10- 
MY10+ 
MY10- 
MY10+ 
Unseparated 
Cells 
Cells 
Cells 
Cells 
__________________________________________________________________________ 
Exp. 1 
Viable cell 100 85 2.1 82 2.5 
recovery (%) 
Colonies/10.sup.5 
CFC-GM 
159 43 3740 11 4190 
cells: BFU-E 95 10 1610 0 1975 
Mixed 6 0 125 0 163 
Exp. 2 
Viable cell 100 96 1.6 
98 1.4 
recovery (%) 
Colonies/10.sup.5 
CFC-GM 
305 93 12000 
43 10750 
cells: BFU-E 130 70 4380 13 2880 
Mixed 0 0 625 2 375 
__________________________________________________________________________ 
EXAMPLE 9 
Isolation of MY10-positive Cells From Marrow Buffy Coat 
The isolation procedure was tried on a buffy coat (rather than a 
Ficoll-Hypaque) preparation of marrow cells. A MY10-positive cell fraction 
could be isolated from marrow buffy coat which was substantially enriched 
in cells with blast cell morphology, the light scattering characteristics 
of MY10-positive cells, and in hematopoietic colony-forming cells. 
However, it was necessary to use a ratio of 1-4 microspheres per nucleated 
cell to efficiently isolate the MY10-positive cells. It was therefore 
decided to use a Ficoll-Hypaque gradient to isolate MMC, since with 
Ficoll-purified cells only 0.5 microsphere per nucleated cell was 
necessary for the isolation procedure. 
EXAMPLE 10 
Chymopapain Treatment of Normal Rat Bone Marrow Cells 
Rat marrow cells were treated with either chymopapain or control medium as 
in Example 2. The treatment had no significant effect on cell viability 
(trypan blue dye exclusion). In addition, no significant injury to rat 
colony-forming cells was detected by in vitro cultures in agar. 
EXAMPLE 11 
Effect of Chymopapain Treatment on Rat Stem Cells Assayed by Marrow 
Transplantation 
Table 3 shows the ability of the treated rat bone marrow cells of Example 
10 to reconstitute hematopoiesis after injection into lethally irradiated, 
syngeneic rats, using as marrow grafts injected cell doses chosen such 
that the cell number would be nearly limiting for engraftment. Experiments 
1 and 2 were performed using grafting cell doses of 1 and 5 million cells 
per rat, and Experiment 3 used 2 and 5 million cells per rat. All animals 
receiving no marrow cell rescue (total body irradiation only) died 12-14 
days after irradiation. These animals had marked pallor prior to death, 
and their bone marrows were extremely hypocellular upon autopsy. 
In contrast, when 5 million treated or control cells were engrafted, all 
animals survived. At a more limiting grafting cell dose of 2 million 
starting cells (based on cell counts prior to chymopapain or sham 
treatment), some animals died, but nearly all lived several days beyond 
the radiation controls; survival was identical in the treated vs. control 
groups. Autopsies of these animals suggested early marrow engraftment, 
with identifiable hematopoietic cells within a hypocellular marrow. 
Similar results were obtained using a dose of 1 million starting cells per 
irradiated rat. Extension of the time of chymopapain incubation with 
marrow cells from 10 to 30 minutes did not decrease the ability of treated 
cells to reconstitute hematopoiesis in these irradiated rats. 
TABLE 3 
__________________________________________________________________________ 
Effect of Chymopapain Treatment on Engraftment of 
Rat Bone Marrow Cells in Lethally Irradiated Rats 
Treatment 
Treatment 
Grafting 
Days of Survival Overall 
(Sham or 
Duration 
Cell Dose 
(for individual rats) 
Survival 
Chymo) (min) (millions) 
Exp. 1 
Exp. 2 
Exp. 3 
(n/total) 
__________________________________________________________________________ 
-- -- None 12,12 13,13 13,14 0/6 
-- -- 1 60+,60+ 
50+,50+ 
ND 4/4 
-- -- 5 60+,60+ 
50+,50+ 
25+,25+ 
6/6 
Sham 10 1 18,18 14,15 ND 0/4 
Chymopapain 
10 1 15,21 13,13 ND 2/8 
60+, 60+ 
15,21 
Sham 10 2 ND ND 19,20 2/4 
25+,25+ 
Chymopapain 
10 2 ND ND 22,25+ 
3/4 
25+,25+ 
Sham 10 5 60+,60+ 
50+,50+ 
ND 4/4 
Chymopapain 
10 5 60+,60+ 
50+,50+ 
25+,25+ 
8/8 
25+,25+ 
Chymopapain 
30 1 15,17 14,15 ND 0/8 
17,18 20,20 
Sham 30 2 ND ND 25+,25+ 
4/4 
25+,25+ 
Chymopapain 
30 2 ND ND 23,25+ 
3/4 
25+,25+ 
Sham 30 5 ND ND 25+,25+ 
4/4 
25+,25+ 
Chymopapain 
30 5 ND ND 25+,25+ 
4/4 
25+,25+ 
__________________________________________________________________________ 
EXAMPLE 12 
Large-Scale Isolation of CD34-Positive Cells From Bone Marrow 
Bone marrow was processed as described in Examples 3 and 4 except that a 
COBE 2991 processing unit was used for the Ficoll processing and washing 
steps, and incubations were carried out in tissue culture flasks of 75 
cm.sup.2 surface area. The amount of MY10 monoclonal antibody used, the 
microsphere:cell ratio and the concentration of chymopapain were the same 
as in the previous Examples. Manual 100-cell differentials were performed 
on cytospins of the isolated cells from two separate isolation runs (Table 
4). The quickness of the manual differential makes it useful for rapidly 
assessing the purity of a cell population. 
The most informative characterization of the cell types present in the 
final, isolated population was provided by the use of direct and indirect 
immunofluorescence assays for cell surface markers. Most of the monoclonal 
antibodies directed against cell surface markers recognize epitopes which 
are not damaged or destroyed by chymopapain as used in the isolation 
procedure. While the epitope recognized by monoclonal MY10 does not remain 
intact after the treatment with chymopapain, other determinants on the 
CD34 glycoprotein do remain intact. TUK3 monoclonal antibody reacts with 
one such determinant and thus allows a direct enumeration of the 
CD34-positive cells present in the final isolated population. A sample of 
the TUK3 antibody was obtained from Dr. Barbara Uchanska-Ziegler, 
Institute fur Experimentelle Imunologie, Universitat Marburg, 
Deutschhausstrasse 1, D-3550 Marburg, Germany. In Isolation 1, 95% of the 
cells reacted with TUK3 while in Isolation 2 only 43% were labelled. 
A variety of other monoclonal antibodies are used to identify cells other 
than CD34-positive cells present in the final isolate. An anti-leucocyte 
FITC (CD45)+Anti-Leu M3 PE (CD14) reagent (LeucoGATE, Becton Dickinson 
Immunocytometry Systems) allows identification of nucleated RBC, mature 
lymphocytes, and monocytes. Anti-Leu 1 (CD5) and Anti-Leu 4(CD3) are used 
to identify T cells while Anti-Leu 12 (CD19) and Anti-Leu 16 (CD20) are 
used as B cell markers. Granulocytes are marked by a monoclonal antibody 
directed against CD15. The information obtained by use of these antibodies 
was analyzed to give the cell distribution by cell surface marker 
presented in Table 4. 
TABLE 4 
______________________________________ 
Composition of Isolated CD34-positive Cell Populations 
Manual Differential 
Cell Surface 
Cell Type % Markers (%) 
______________________________________ 
Isolation 1 
Blasts 85 95 
Nucleated RBC 
3 1 
Lymphocytes 0 0.5 
Granulocytes 12 3.5 
Isolation 2 
Blasts 36 43 
Nucleated RBC 
48 32 
Lymphocytes 7 18 
Granulocytes 9 5 
______________________________________ 
EXAMPLE 13 
Light Scattering Characteristics of Isolated Bone Marrow Cells 
Plots of forward vs. side scatter were obtained for the three cell 
populations produced in the isolations of Example 12: 1) unseparated, 
Ficoll-processed cells; 2) cells which did not bind to microspheres and 
are therefore depleted of MY10-positive cells; and 3) the cells isolated 
after chymopapain treatment of cell-microsphere complexes which include 
the MY10-positive cells. MY10-positive cells have light scattering 
properties characteristic of the "BLAST" and "LYMPH" windows. The third 
window is the "GRAN" window. The percent of each cell population occurring 
in each of these windows is reported in Table 5. In both Isolations 1 and 
2, unseparated cells and the MY10-depleted cells have similar light 
scattering profiles and similar cell distributions among the three 
windows. The isolated cells, as expected, occur predominantly in the 
"LYMPH" and "BLAST" windows, with the marked increase in the percentage of 
cells in the "BLAST" window being particularly indicative of the 
enrichment for MY10-positive cells. 
In Isolation 2, only approximately 43% of the cells were CD34-positive as 
determined by cell surface markers (see Example 12). The major 
contaminating cell types, nucleated red blood cells and lymphocytes, also 
tend to have light scatter properties characteristic of the "LYMPH" window 
with some spillage into the "BLAST" window. Light scatter is therefore 
most useful for its indication of depletion of granulocytes and monocytes 
and for its indication of enrichment of cells occurring in the "BLAST" 
window which are predominantly CD34-positive cells. 
TABLE 5 
______________________________________ 
Distribution of Cells by Light Scatter 
Cell Population 
Unseparated MY10 Depleted 
Isolated 
Window (%) (%) (%) 
______________________________________ 
Isolation 1 
Lymphocyte 
22 22 23 
Blast 9 8 62 
Granulocyte 
38 39 3 
Isolation 2 
Lymphocyte 
23 20 27 
Blast 10 9 50 
Granulocyte 
43 46 4 
______________________________________ 
EXAMPLE 14 
Hematopoietic Colony-Forming Capacity 
The final CD34-positive cell fractions obtained in Example 12 were tested 
for the enrichment of colony-forming cells relative to the starting and 
MY10-depleted cell populations. Colony forming data are presented in Table 
6. 
TABLE 6 
__________________________________________________________________________ 
Colony-Forming Capacity of Cell Fractions 
Unseparated 
Unbound Cells 
Isolated Cells 
Cells (MY10-) (MY10+) 
__________________________________________________________________________ 
Isolation 1 
Viable cell 100 59 .76 
recovery (%) 
Colonies/10.sup.5 
CFC-GM 
370 18 7200 
cells: BFU-E 186 8 1950 
Day 14 Blasts 
5.8 0 47.5 
Isolation 2 
Viable cell 100 80 2.36 
recovery (%) 
Colonies/10.sup.5 
CFC-GM 
143 15 18625 
cells: BFU-E 75 2.5 6425 
__________________________________________________________________________ 
In both isolations the final cell fraction was highly enriched for 
colony-forming cells. Purified MY10 cells in this assay system typically 
give rise to 5,000 to 10,000 colonies per 10.sup.5 cells planted. Thus the 
functional capacity of the CD34-positive cells to form colonies does not 
appear to be impaired by the isolation procedure, including treatment with 
chymopapain. Furthermore, few colony-forming cells remained in the 
unbound, MY10-depleted cell fraction.