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Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1820-1827
By
From the Department of Hematology/Oncology, University Medical
Center, Freiburg; and Institute for Experimental Cancer Research, Tumor
Biology Center, Freiburg, Germany.
Tumor cells have been found in autologous hematopoietic cell
transplants used after high-dose chemotherapy. To specifically eliminate contaminating mammary tumor cells during ex vivo expansion of
CD34+ hematopoietic progenitor cells, we used recombinant
immunotoxins (ITs) directed against cell-surface antigens expressed on
mammary carcinoma cells. ITs were expressed from fusion cDNAs combining a single-chain antibody fragment (scFv) directed against the Erb-B2 or
epidermal growth factor (EGF) receptors with a truncated
Pseudomonas exotoxin A fragment devoid of its cell-binding
domain. CD34+ hematopoietic progenitor cells did not
express Erb-B2 and EGF receptors as detected by Western blotting. Ex
vivo expansion of total hematopoietic cells or of colony-forming cells
from CD34+ progenitors in the presence of stem-cell
factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin
(Epo) was not affected when ITs were added to the cultures. In
contrast, MDA-MB 453 and MCF-7 mammary carcinoma cells were depleted in
a dose- and time-dependent manner by more than 3 log in coculture with
CD34+ cells over a period of 7 days in the presence of
100 to 1,000 ng/mL of anti-Erb-B2 IT. This included elimination of the
subpopulations with regrowth potential. Similarly, addition of either
anti-Erb-B2 or anti-EGF receptor ITs to primary breast cancer cells
isolated from patients with metastatic disease resulted in elimination of cytokeratin-positive cells in seven of seven samples. ITs are highly
efficient and convenient to use for the depletion of mammary tumor
cells during ex vivo expansion of hematopoietic progenitor-cell autografts.
HIGH-DOSE CHEMOTHERAPY in combination
with the transplantation of autologous hematopoietic progenitor cells
is being used for the treatment of hematologic malignancies and a
variety of solid tumors.1 Clinical trials using high-dose
chemotherapy protocols in patients with breast cancer have suggested an
increased therapeutic benefit when compared with conventional
chemotherapy regimens. This includes increased quality of life,
improved response rates, and prolonged survival.2,3
However, tumor-cell contamination of autologous grafts has been
demonstrated in various malignancies and could contribute to disease
relapse. This was shown by genetic marking of tumor cells within the
graft.4-6 Tumor cells have been found at an incidence of up
to one per 104 cells in bone marrow, and at approximately
one per 105 cells within mobilized peripheral blood
harvests of breast cancer patients.7,8 An indication that
reinfused contaminating breast cancer cells may contribute to disease
relapse in breast cancer comes from experiments showing that mammary
tumor cells residing in bone marrow are capable of in vitro
growth.7,9 In addition, reports on breast cancer patients
and patients with gynecologic cancer suggest a positive association
between numbers of tumor cells reinfused after transplant and adverse
clinical outcome.10-13
Enrichment of CD34+ hematopoietic progenitor cells from
leukapheresis products has been shown to reduce tumor-cell numbers in
transplants.14 However, large-scale selection of
CD34+ cells has been found less efficient compared with
that found in laboratory scale experiments, with purities of 50% to
85%. Residual tumor cells have been demonstrated in autologous grafts after CD34+ selection15 (and our own
unpublished results). Therefore, additional methods have been
investigated to achieve tumor-cell-free grafts, such as ex vivo
culture and expansion of hematopoietic progenitor cells.16
For ex vivo expansion, hematopoietic cells are seeded into
culture medium in the presence of a combination of hematopoietic growth
factors, resulting in 100- to 1,000-fold amplification of total cell
numbers and up to 100-fold amplification of lineage-committed colony-forming progenitors.16-19 More primitive
hematopoietic stem-cell populations, eg, long-term bone marrow
culture-initiating cells (LTCIC) have been found to be maintained at
input numbers or to amplify to a limited degree during ex vivo
expansion.19,20 CD34+ blood progenitor cells
expanded ex vivo in the presence of stem-cell factor (SCF), interleukin
(IL)-1 Immunotoxins (ITs) were initially developed for the elimination of
lymphocytes from allogeneic bone marrow transplants23,24 by
conjugating potent toxins of plant or bacterial origin to monoclonal antibodies, or recombinant antibody variant chain domains, which specifically target surface antigens. ITs that target epithelial cells
of certain tumor-cell types have subsequently been
constructed.25-27 In this study, we used single-chain
antibody fragment (scFv) directed against the Erb-B2 receptor or
against the epidermal growth factor (EGF) receptor genetically fused to
a modified Pseudomonas exotoxin A.27,28
Pseudomonas exotoxin A consists of several functional domains
that are responsible for the binding to the surface of mammalian cells,
uptake and translocation to the cytosol, and catalytic activity. Upon
cell binding and internalization, exotoxin A is cleaved by a cellular
protease within its domain II and a N-terminal 28-kD and a C-terminal
37-kD fragment are generated. After reduction of a disulfide bond, the
C-terminal enzymatically active fragment translocates to the cytoplasm.
The catalytic domain of exotoxin A adenosine diphosphate
(ADP)-ribosylates and inactivates eukaryotic elongation factor 2, an
essential component in protein synthesis.29 Both the Erb-B2
and EGF receptor have been found to be overexpressed in a high
percentage of mammary-cell carcinomas.30 This study
investigated the potential of these ITs to deplete autologous stem-cell
grafts from contaminating tumor cells during ex vivo culture. We found
that ITs directed against Erb-B2 and EGF receptors spare hematopoietic
cells, and at the same time deplete tumor cells from cocultures with
CD34+ cells by 3 log within a period of 5 to 7 days. ITs
are potent tools for purging breast cancer cells from hematopoietic
cell populations.
Breast cancer cells.
Breast cancer cell lines MDA-MB-453 and MCF-7 were obtained from the
American Type Culture Collection (ATCC, Rockville, MD) and
grown in Dulbecco's modified minimal essential medium (DMEM; BioWhittaker, Verviers, Belgium) supplemented with
L-glutamine and 10% fetal calf serum (FCS; Pan Systems,
Passau, Germany). Primary breast cancer cells were isolated from bone
marrow samples, ascites, or pleural effusions from patients with
metastatic disease after informed consent. Cells were washed in
phosphate-buffered saline (PBS) and subsequently cultured in
Expression and purification of ITs.
The genes that encode the ErbB2 and the EGF receptor specific
recombinant single-chain antibody toxins scFv(FRP5)-ETA and scFv(14E1)-ETA were constructed by fusing a truncated Pseudomonas aeruginosa exotoxin A gene (ETA) to the respective scFv gene
derived from the genes that encode the light- and heavy-chain variable domains of the monoclonal antibodies FRP5 and 14E1, respectively, as
described previously.27,28 Briefly, the resulting plasmids were grown in Escherichia coli strain HB 101 and fusion
proteins were purified from inclusion bodies on Ni2+ loaded
chelating sepharose columns (Pharmacia, Uppsala, Sweden), eluted in two steps with 50 mmol/L and 150 mmol/L imidazole in solubilization buffer, respectively, and dialyzed overnight against PBS
containing 400 mmol/L arginine followed by dialysis against PBS.
Purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and quantitated by densitometry after
Coomassie-blue staining in comparison to bovine serum albumin (BSA)
standards.
Detection of Erb-B2 and EGF receptor expression by immunoblotting.
Human tumor cells and purified human CD34+ cells were
extracted in lysis buffer (50 mmol/L Tris-Cl, pH 7; 5 mmol/L EDTA; 1% Triton X-100; 150 mmol/L NaCl; 1 mmol/L phenylmethylsulfonyl fluoride [PMSF]; 80 µg/mL aprotinin, 50 µg/mL leupeptin; 4 µg/mL
pepstatin). A431 and SKBR3 cells from ATCC were used as a positive
control. Extracts were subjected to a 7.5% SDS-PAGE and proteins were
electroblotted onto nylon membranes. The p185Erb-B2 and p170 EGF
receptor proteins were detected using the 21N and 12E antisera,
respectively27,28 and an enhanced chemiluminescence
detection kit (Amersham, Braunschweig, Germany).
CD34+ blood progenitor-cell selection and
expansion.
Peripheral blood progenitor cells were obtained from patients with
solid tumors (two with bronchial carcinoma, two with mammary carcinoma)
or lymphomas (two) after informed consent. Mobilization of progenitors
into blood was induced by a 1-day course of standard-dose chemotherapy
that included etoposide, ifosfamide, cisplatin, and epirubicin, and
subsequent daily application of granulocyte colony-stimulating factor
(G-CSF). Aphereses were performed using Baxter CS3000 cell separators (Baxter, Round Lake, IL) on day 10 to 12 after the start of
mobilization. CD34+ cells were selected using Ceprate LC
columns (CellPro, Bothell, WA) according to the manufacturer's
instructions. The purity of the CD34+-enriched cells was
analyzed by flow cytometry on a FACScan (Becton Dickinson, Heidelberg,
Germany) using phycoerythrin-conjugated monoclonal HPCA-2 anti-CD34
antibody (Becton Dickinson). Expansion cultures were performed in
24-well plates at 37°C in a 5% CO2 atmosphere using RPMI
1640 medium (BioWhittaker, Brussels, Belgium) that contained 10% FCS
and 3 ng/mL IL-1 Coculture of CD34+ progenitor cells and breast
cancer cells.
Tumor cells (MDA-MB-453 and MCF-7) were mixed with 3 × 104 CD34+ blood progenitor cells at a ratio of
tumor cells to CD34+ cells of 1:10, cultured in 1 mL IMDM,
10% FCS, 3 ng/mL IL-1 Effect of ITs on primary breast cancer cells.
Enriched primary breast cancer cells (>60% cytokeratin-positive
cells) isolated from the patient samples as described earlier were
seeded into 24-well plates in FCS-supplemented Tumor-cell clonogenic assay.
The number of viable clonogenic tumor cells after IT treatment was
assessed in limiting dilution clonogenic assays. MDA-MB-453 and MCF-7
cells were seeded into 24-well plates (2 × 104/well)
and treated with different concentrations (up to 1,000 ng/mL) of ITs
for a 4-day period. The supernatant was then removed and the adherent
cells were detached with trypsin-EDTA and subsequently reseeded in 100 µL of normal growth medium without addition of ITs in 96-well plates
via the automatic cell deposition unit of a MoFlo flow cytometer-cell
sorter (Cytomation, Fort Collins, CO). Series of 1, 10, and 30 cells/well were prepared. Only events residing in the viable cell
gates, as assessed by the forward and 90° light scatter of the cells,
were sorted. The microtiter plates were placed in a humidified
incubator with 5% CO2 at 37°C for 30 days. To avoid a
change in the osmolarity of the medium due to the 4-week incubation
time, the plates were sealed in gas-permeable polyethylene bags and
only the 60 inside wells of each plate were used, while the outer wells
were filled with H2O. Wells that contained at least one
cluster of greater than 30 cells, as assessed by visual inspection
under an inverted microscope, were scored as positive.
Cytokeratin staining.
Cells from the mixed cultures and the primary cultures were washed
twice in hydroxyethyl-piperazine-ethane sulfonic acid (HEPES) buffer
and attached to Poly-L-lysine-coated adhesion slides
(BioRad, Munich, Germany) consisting of 12 spots of 5 mm diameter.
Duplicate analyses were performed from each culture. Cells were
anchored and dried on the spots according to the manufacturer's
recommendations and slides were stored in sealed plastic bags at
Expression of Erb-B2 and EGF receptor on CD34+ blood
progenitor cells and mammary carcinoma cells.
The basis of specific tumor-cell targeting is the selective recognition
of cell-surface epitopes that mediate binding of ITs and their transfer
into malignant cells. Strong expression of EGF receptor was detected in
A431 control cells, but EGF receptor was undetectable in
CD34+ cells (five of five patient samples). Similarly,
Erb-B2 receptor was found to be expressed at various levels in cell
lines as a 185-kD protein, but was undetectable in CD34+
hematopoietic cells (four of four patient samples). In patient tumor
samples, EGF receptor was found to be expressed in five of seven
specimens, and Erb-B2 receptor in five of six samples. Representative
analyses of CD34+ cell samples and patient tumor samples
for Erb-B2 and EGF receptor expression are shown in Fig
1. These data demonstrate that target proteins for ITs are selectively expressed on mammary tumor cells.
Effect of ITs on development and ex vivo expansion of
CD34+ blood progenitor cells.
Ex vivo culture of hematopoietic progenitors aims at growth
factor-mediated cell amplification, and involves the generation of
colony-forming progenitor cells. In direct methylcellulose cultures of
CD34+ cells, ITs showed no detectable direct effects on the
development of both colony-forming units granulocyte-macrophage
(CFU-GM) and erythroid burst-forming units (BFU-E) at IT concentrations
as high as 1,000 ng/mL (Fig 2). Moreover,
ex vivo expansion of total nucleated cells and of colony-forming cells
from CD34+ progenitor cells in the presence of SCF,
IL-1
Effect of ITs on tumor-cell elimination during ex vivo expansion of
CD34+ progenitor cells.
There are only few breast cancer cells within
CD34+-mobilized blood specimens.15 Therefore,
to assess the optimal conditions for an effective ex vivo purging
procedure, we performed mixing experiments with tumor-cell lines added
to CD34+ progenitor cell cultures. MDA-MB 453 or MCF-7
cells and CD34+ cells were cocultured at a ratio of 1:10.
The cells were kept in medium that corresponded to established ex vivo
expansion conditions in the presence of SCF, IL-1
Elimination of clonogenic tumor cells by ITs.
It is possible that the sensitivity of detection of our
immunohistochemical assay misses small amounts of tumor cells. Tumor cells may include resting cells with high proliferative potential, which may preferentially survive exposure against ITs. For these reasons, we performed tumor-cell regrowth assays. After exposure of
MCF-7 and MDA-MB 453 cells to anti-Erb-B2 IT for 4 days, surviving cells were reseeded into optimal tumor-cell growth medium. Table 1 shows that treatment with anti-Erb-B2 IT
decreased the subpopulations of MCF-7 and MDA-MB 453 cells with
regrowth potential. This indicates that the elimination of tumor cells
by ITs during ex vivo expansion includes the clonogenic populations.
Effect of IT on primary breast cancer cells isolated from patients.
Having established time periods and IT concentrations needed for
effective tumor-cell purging by ITs, we investigated the effect of ITs
against primary breast cancer cells. Pleural effusion, ascites, and
bone marrow aspirates, containing numerous tumor cells, were exposed to
1,000 ng/mL of ITs. The effect of ITs on enriched primary tumor-cell
populations is shown in Table 2. Within a
period of 7 days, viable tumor cells were depleted in three of five
samples expressing Erb-B2 receptor by anti-Erb-B2 IT, or in five of
five samples expressing EGF receptor by anti-EGF receptor IT. In one
case with no detectable expression of EGF receptor, anti-EGF receptor
IT-mediated killing activity was also recorded. Identical results were
observed in the additional presence of hematopoietic growth factors in
the cultures. Therefore, these factors do not influence the efficacy of
ITs in eliminating tumor cells (data not shown). An example of primary
mammary tumor-cell elimination by anti-EGF receptor IT is shown in Fig
5B. These results indicate that ITs can exert their cytotoxic
activities on primary tumor-cell material.
Breast cancer is one of the most common disease entities in medical
oncology. The proportion of cases diagnosed early and treated at a
state of limited disease has increased in recent years. In addition,
the treatment of breast cancer patients with high-dose chemotherapy has
resulted in improved survival rates compared with conventional
treatment regimens.2,3 Contamination of autologous
hematopoietic cell transplants with tumor cells is of clinical
disadvantage.4-6 Cell-separation methods have been
investigated for the ex vivo purging of autologous transplants, exploiting cell-surface markers and monoclonal antibody
technology.14 In contrast to these selection protocols,
which last up to several hours, longer-term purging procedures require
specific culture conditions to ensure the viability or expansion of
hematopoietic progenitor cells. To this end, recombinant hematopoietic
cytokines have been used to maintain and expand hematopoietic
progenitors in culture.16 A minimum number of tumor cells
within a transplant that could affect the course of the disease has not
been defined. Current concepts aim at maximal depletion of transplants
from contaminating tumor cells. A specific molecular targeting of solid tumor cells, eg, by ITs directed specifically against epithelial or
breast cancer cell antigens, is therefore highly desirable for clinical
practice. ITs could be useful in tumor cell purging of hematopoietic
stem cells used as autologous grafts after high dose chemotherapy.
ITs are effective during ex vivo expansion of CD34+
progenitor cells for tumor-cell purging.
The specific activity of ITs may depend on properties related to the
antibodies used, the choice of toxins, the method by which the toxins
are linked to the antibodies, the concentration of the ITs, and the
treatment period. Our results indicate that the antibody
Pseudomonas exotoxin A fusion proteins specific for Erb-B2 and
EGF receptor are not cytotoxic for hematopoietic cells at
concentrations of up to 1,000 ng/mL, including CD34+
hematopoietic progenitors. A number of ITs containing the
Pseudomonas exotoxin,25,26 the toxin
ricin,31 or the recombinant ricin A chain32 and
directed against epithelial antigens have been used to purge bone
marrow from breast cancer cells. Only insignificant toxicity to
hematopoietic progenitors was shown, but a reduction of colony-forming
cells by up to 50% was reported during short incubation times (up to 2 hours).26 We show that treatment periods up to 14 days with
recombinant ITs do not alter the survival and the proliferative
potential of the CD34+ progenitor cells in ex vivo
expansion cultures. We also show that whereas prolonged exposure times
do not result in increasing toxicity to hematopoietic cells, they may
increase the efficiency to eliminate mammary carcinoma cells from
autologous grafts. The extended culture of hematopoietic grafts may
offer advantages of more efficient tumor-cell elimination at a given IT
concentration with no detectable side effects on hematopoietic cells.
Short-term treatments with ITs at concentrations noncytotoxic for
hematopoietic cells have been shown to effectively purge autologous
grafts in previous studies.25,26,33,34 However, these
results must be interpreted with caution, since they refer to removal
of cell lines that show high target antigen expression and rapid IT
internalization. To our knowledge, this is the first report to
demonstrate potent in vitro cytotoxic activity of immunotoxins not only
towards cell lines, but also towards primary breast cancer cells.
Erb-B2 and EGF receptor as targets for purging protocols in breast
cancer.
For antibody-depletion systems to be effective, the antigens recognized
by the ITs must be expressed on the target cells. The antigenic
phenotype of contaminating mammary tumor cells in autologous
hematopoietic transplants is, up to now, poorly characterized. Mapara
et al15 showed that six of nine breast cancer patients with
immunocytochemically cytokeratin-positive CD34+ specimens
were also positive in an EGF receptor-specific reverse transcriptase
(RT)-PCR assay. Moreover, Pantel et al35 showed that 23 of
23 of M1 and 25 of 48 M0 stage breast cancer patients expressed the
Erb-B2 oncogene in bone marrow cytokeratin-positive cells, although
only 10% to 30% of patients express Erb-B2 in their primary tumors.
Therefore, Erb-B2 and EGF receptors are suitable targets for purging
procedures in a majority of breast cancer patient samples.
Immunotoxins as part of integrated purging strategies.
The high efficiency and specificity of ITs against breast cancer cells
makes them a suitable purging tool. ITs might be used in combination
with other established procedures. If several purging steps are
combined, recovery rates of hematopoietic progenitor cells within each
single step are crucial for the success of the manipulation procedures.
Since IT-mediated tumor-cell purging results in no significant loss of
hematopoietic progenitors, it is expected to be superior to
immunomagnetic or immunoaffinity cell-selection procedures. These may
result in an overall stem-cell loss of up to 50%. The superiority of
immunotoxins to drug treatments such as
4-hydroperoxycyclophosphamide36 for ex vivo purging protocols is based not only on their high specificity, but also on the
fact that toxins work by inhibiting protein synthesis and can kill
nondividing cells, whereas most chemotherapeutic agents act by
interfering with DNA synthesis and cell division.
We thank Prof Dr Roland Mertelsmann for the incentive to perform
this cooperative study and for his continuous support. We also thank Dr
Jürgen Finke for valuable suggestions.
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Abstract
Introduction
Abstract
, IL-3, IL-6, and erythropoeitin (Epo) have been used to
mediate hematopoietic reconstitution after high-dose chemotherapy in
patients with solid tumors.
-MEM (GIBCO-BRL, Paisley, Scotland) and 10% FCS for up
to 7 days before use. Cell populations isolated with this method
contained greater than 60% cytokeratin-positive cells, as identified
by immunostaining, and were used as a source of cells for the
evaluation of IT treatment on primary breast cancer cells. Cells were
detached with trypsin-ethylenediamine tetraacetic acid (EDTA) solution
(Sigma, Munich, Germany) immediately before use.
20°C until use. For cytokeratin staining, cells were fixed in
serial dilutions of aceton in ethanol for 5 minutes at 4°C and
immunostained in an alkaline phosphatase-antialkaline phosphatase
assay (DAKO, Hamburg, Germany).
) and BFU-E
numbers (
) in a light microscope on day 14. Values are the means ± SD of duplicate determinations. Results shown are representative of at
least four experiments in each case (ie, anti-Erb-B2 and anti-EGF
receptor IT) with CD34+ cells derived from different
patients.
) and cytokeratin-negative cells (
A novel, highly potent antibody-toxin specific for the EGF receptor.
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75:1575,
1997