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Blood, Vol. 92 No. 2 (July 15), 1998:
pp. 589-595
Malignant Progenitors From Patients With Acute Myelogenous
Leukemia Are Sensitive to a Diphtheria
Toxin-Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein
By
Donna E. Hogge,
Cheryl L. Willman,
Robert J. Kreitman,
Marc Berger,
Philip D. Hall,
Kenneth J. Kopecky,
Chris McLain,
Edward P. Tagge,
Connie J. Eaves, and
Arthur E. Frankel
From the Terry Fox Laboratory, British Columbia Cancer Agency,
Vancouver, British Columbia, Canada; the Department of Pathology,
University of New Mexico Cancer Center, Albuquerque, NM; the Laboratory
of Molecular Biology, National Cancer Institute, National Institutes of
Health, Bethesda, MD; the Departments of Surgery and Pharmaceutical
Sciences, Medical University of South Carolina, Charleston, SC; the
SWOG Statistical Center, Fred Hutchinson Cancer Research Center,
Seattle, WA; and the Comprehensive Cancer Center at Wake Forest,
Winston Salem, NC.
 |
ABSTRACT |
We have previously demonstrated that human granulocyte-macrophage
colony-stimulating factor (GM-CSF) fused to a truncated diphtheria
toxin (DT388-GMCSF) kills acute myelogenous leukemia (AML) cell lines
bearing the GM-CSF receptor. We now report that exposure of malignant
cells from 50 different patients with AML for 48 hours in culture to
DT388-GMCSF reduces by a median of 1.6 logs (range, 0 to 3.7 logs) the
number of leukemic cells capable of forming colonies in semisolid media
(leukemic colony-forming cells [CFU-L]) with a median
IC50 of 3 × 10 12 mol/L (range, 5 to
>4,000 × 1012 mol/L). Furthermore, the cell kill is
dependent on the presence of high-affinity GM-CSF receptors on leukemic
blasts, because CFU-L from 27 of 28 AML samples expressing  35 GM-CSF
receptors per cell were inhibited by the toxin, whereas the colony
growth from all 4 leukemic samples (2 AML, 1 acute lymphoblastic
leukemia [ALL], and 1 prolymphocytic leukemia [PLL]) that had less
than 35 receptors per cell was unaffected by the drug. Sensitivity of
CFU-L to DT388-GMCSF was seen regardless of the clinical responsiveness of the patient's leukemia to standard chemotherapy agents. In contrast, clonogenic cells from normal bone marrow formed colonies at
near control numbers after exposure to much higher toxin concentrations (4 × 109 mol/L) than those required to kill CFU-L from
most patients. Thus, leukemic progenitors isolated directly from the
peripheral blood of most AML patients show the same sensitivity to
DT388-GMCSF as previously demonstrated for AML cell lines. Under the
same conditions of exposure, normal hematopoietic progenitors are
relatively unaffected by DT388-GMCSF, suggesting its potential as a
therapeutic agent in AML.
| |
INTRODUCTION |
ACUTE MYELOGENOUS leukemia (AML), the
most common acute leukemia in adults, is associated with a 70%
complete remission (CR) rate after standard induction
chemotherapy regimens.1 Intensive postremission therapy in
combination with allogeneic bone marrow transplantation offers the
possibility of cure to some of these individuals; however, fewer than
20% of all patients with the diagnosis of AML will have prolonged
disease-free survival.2 Resistance to standard
chemotherapeutic drugs is an important cause of the relapsed,
refractory leukemia to which most patients succumb.3
A number of identified drug resistance phenotypes are due to
overexpression of specific proteins, and the concentration of these
molecules in leukemic blasts has been correlated with response to
cytotoxic chemotherapy.4-8 In several cases, the resistance protein transports or inactivates xenobiotics such as anthracyclines. Pharmacologic reversal of MDR-1-related anthracycline resistance has,
to date, been associated with toxicities to marrow,
gastrointestinal tract, and the central nervous system;
altered anthracycline pharmacodynamics; and minimal improvements in
response rate or disease-free survival.9 An
antibody-targeted cytotoxic drug, anti-CD33-calicheamicin, was recently
tested in a clinical trial and had reduced side effects.10 However, most patients rapidly developed resistance due to active drug
efflux. Thus, novel agents that are cytotoxic to leukemic blasts and
bypass multidrug resistance phenotypes are urgently needed.
One such class of therapeutics are protein toxins covalently linked to
peptide ligands. The ligand directs the molecule to the surface of
specific cell types. The toxin moiety then enters the cell and
catalytically inactivates protein synthesis. Toxins constructed that
target AML blasts include the following: anti-CD33-blocked ricin,
anti-CD33-gelonin, diphtheria toxin-interleukin-3 (IL-3), anti-transferrin receptor-ricin A chain, granulocyte-macrophage colony-stimulating factor (GM-CSF)-ricin, GM-CSF-Pseudomonas exotoxin, and diphtheria toxin-GM-CSF.11-21 Each of these reagents
inhibited protein synthesis by 50% (IC50) in cell lines in
a dose- and time-dependent manner. The least active drugs were
anti-transferrin receptor-ricin A chain, GM-CSF-ricin, and
GM-CSF-Pseudomonas exotoxin, with IC50s of approximately 3 × 1010 mol/L. Anti-CD33-blocked ricin and
anti-CD33-gelonin had intermediate IC50s of
1010 mol/L, and diphtheria toxin-IL3 and diphtheria
toxin-GM-CSF produced IC50s of 3 × 10-11
mol/L. The variability in sensitivity that AML cell lines show to
targeted toxins has been attributed to premature intracellular routing
of ricin, ricin A chain, gelonin, and Pseudomonas exotoxin conjugates
to lysomes. In contrast, the diphtheria toxin fusion proteins are able
to translocate to the cytosol from a prelysosomal intracellular
compartment. Specificity of cell kill has been demonstrated for several
of these conjugates, including anti-CD33-blocked ricin, anti-CD33-gelonin, diphtheria toxin-IL3, and diphtheria toxin-GM-CSF. Reductions in normal human or murine marrow progenitors were seen after
toxin conjugate exposure, but, in each case, the effect was much
smaller than seen with AML cell lines.17,19,20
A critical question is whether these chimeric proteins, which require
functional cell surface receptors, intact intracellular routing
pathways, and sensitive protein synthesis machinery for intoxication,
will kill malignant cells isolated directly from patients with
leukemia. With several previously studied lymphoid malignancy-targeted
toxins, including anti-CD5-ricin A chain and anti-CD25
(sFv)-Pseudomonas exotoxin, fresh leukemia cells were much less
sensitive than cell lines.22,23 Cytotoxicity was enhanced
for anti-CD5-ricin A chain by adding the monocarboxylic acid ionophore,
monensin, and for anti-CD25-Pseudomonas exotoxin by prolonged
incubation. Both of these interventions improve intracellular toxin
transport to compartments optimal for translocation to the cytosol. Few
similar experiments have been conducted with toxins targeted to myeloid
leukemia.
Roy et al11 tested the cytotoxicity of anti-CD33-blocked
ricin to leukemic colony-forming cells (CFU-L) from 12 AML patients and
found that reductions in CFU-L colony formation were dose- and
time-dependent. Perentesis et al21 observed a 1 to 3 log kill of CFU-L from 7 of 9 therapy-refractory AML patients after exposure to diphtheria toxin-GM-CSF. However, it is possible that some
of the observed effects in the latter studies were due to the presence
of GM-CSF in the control but not in the toxin-treated test cultures.
To perform a comprehensive study of the sensitivity of leukemic
clonogenic cells to diphtheria toxin-GM-CSF, we collected a series of
50 AML samples representing every French-American-British (FAB) subtype
except M3 and M6. These included samples from patients who were known
at the time to have disease both sensitive to and refractory to
conventional cytotoxic agents. Also included were several samples from
patients with lymphoid leukemias and normal bone marrow controls. We
determined the GM-CSF receptor density and kd for the
malignant blasts from each of the malignant samples and then assessed
the ability of DT388-GMCSF to inhibit the growth of normal and leukemic
clonogenic cells in standard semisolid growth factor-supplemented
medium. As expected, some of the (untreated) leukemic cell samples
failed to yield any discrete colonies in this assay. Nevertheless, for
33 of the 53 samples, we were able to determine for the toxin both the
log kill and IC50 of CFU-L. The results demonstrate that
leukemic progenitors from the majority of primary human AML patients
are sensitive to the cytotoxic effects of this conjugate, regardless of
their responsiveness to conventional chemotherapy drugs, whereas normal
clonogenic cells largely are not.
| |
MATERIALS AND METHODS |
Cells.
Heparinized peripheral blood samples from 50 patients with a diagnosis
of AML and 3 patients with lymphoid leukemias and normal bone marrow
samples from 4 individuals donating marrow for allogeneic transplantation were obtained after informed consent was obtained (MUSC
protocol #7123, Terry Fox Laboratory protocol #C96-0429, and SWOG
protocol #8600). Low-density (<1.077 g/mL) cells were isolated using
a Ficoll-Hypaque gradient.
Freshly isolated or thawed, cryopreserved leukemic samples were
suspended in RPMI 1640 medium (Irvine Scientific, Santa Ana, CA) with
15% fetal calf serum (FCS). After incubation for 1 hour at
37°C/5% CO2 in 75-cm2 tissue culture
flasks (Costar Scientific Corp, Cambridge, MA), the nonadherent cells
were collected and mixed with 0.1 mL anti-CD2 immunobeads (Dynal, Oslo,
Norway). The bead-cell mixture was gently rocked at 4°C for 30 minutes, and then CD2+ cells were depleted by magnetic
separation. The procedure removed greater than 95% of CD2+
T cells from initial preparations based on flow cytometry using anti-CD3-phycoerythrin binding (data not shown). Cells were then counted and aliquoted for the GM-CSF receptor and DT388-GMCSF sensitivity studies described below.
Fusion toxin.
DT388-GMCSF was prepared and purified as previously
described.17 Material was stored as aliquots at 840 µg/mL
in phosphate-buffered saline (PBS) plus 1% human serum albumin at
 20°C until used. The material used in this study was found
to kill the HL60 human AML cell line with an IC50 of 2 × 1012 mol/L in a 48-hour
3H-thymidine incorporation assay and to produce a maximum
3.5 log depletion of HL60 cells forming colonies in semisolid
medium.15
GM-CSF receptor density.
Aliquots of 1 to 6 × 106 cells in RPMI 1640 plus
2.5% bovine serum albumin and 20 mmol/L HEPES and 0.2% sodium azide
were mixed with varying amounts of 125I
Bolton-Hunter-labeled human GM-CSF (80 to 120 µCi/µg; NEX249; DuPont, Boston, MA) with or without excess (1,500 ng) cold GM-CSF (Immunex, Seattle, WA) in a total volume of 150 µL in 1.5-mL
Eppendorf tubes. Cells were incubated at 37°C for 30 minutes and
then layered over a 200 µL oil phthalate mixture (1 part
dioctylphthalate and 1.5 parts dibutylphthalate; Aldrich, Milwaukee,
WI). After centrifugation at 12,000 rpm for 1 minute in a microfuge at
room temperature, both pellets and supernatants were saved and counted
in an LKB-Wallac 1260 Multi-gamma counter (Gaithersburg,
MD) gated for 125I with 50% counting efficiency.
Background cpm was calculated by linear extrapolation from incubations
with excess cold GM-CSF. Scatchard plots of specific bound/free versus
specific bound cpm were made. Receptor number/cell was calculated using
the following equation: the value for the x intercept / (specific
activity in µCi/µg × the cell number × [4.2 × 108]). kd was calculated as the x intercept times
2.7 × 1013 divided by the y intercept times
the specific activity. The Statistical software package (Statsoft,
Tulsa, OK) was used for linear regression with separate analysis of the
6 lowest concentration 125I-GM-CSF points and the 4 highest
concentration 125I-GM-CSF points. Nonlinear regression
using the Radlig program (Biosoft) confirmed estimates from Scatchard
plots for 4 samples. Receptor densities of 0 were recorded based on the
absence of specific 125I binding or negative values for kd.
Sensitivity of CFU-L and normal bone marrow clonogenic cells
(colony-forming cells [CFC]) to DT388-GMCSF.
Sensitivities of fresh leukemic blast progenitors and normal bone
marrow CFC to DT388-GMCSF were tested in suspension culture. Aliquots
of 106 AML blasts or light density marrow cells were placed
in suspension culture with different concentrations of DT388-GMCSF (0 to 4 × 109 mol/L) in 24-well flat-bottomed
Costar plates. AML cells were cultured in 1 mL of RPMI 1640 with 20%
FCS and 50 ng/mL granulocyte colony-stimulating factor (G-CSF; Amgen,
Thousand Oaks, CA) plus 1 of 10 different toxin concentrations over the
indicated range. Normal marrow cells were cultured with 1 of 4 different DT388-GMCSF concentrations over the same range in 0.5 mL
serum-free medium (StemCell Technologies, Vancouver, British Columbia,
Canada) without added cytokines. Suspension cultures without the fusion
toxin but the same in all other respects served as the untreated
controls for determining the percentage of survival of clonogenic cells from both leukemic and normal samples.
CFU-L assays.
After 48 hours of incubation at 37°C in 5% CO2, 100 µL from each AML suspension culture containing different
concentrations of DT388-GMCSF was mixed with 3 mL RPMI + 15% FCS plus
50 ng/mL G-CSF and GM-CSF and 0.3% agarose (SeaPlaque; FMC
Bioproducts, Rockland, ME) and poured into 35-mm gridded petri dishes
(Nunc, Naperville, IL). In some experiments, 10% medium conditioned by the 5637 human bladder carcinoma cell line was added in the agarose colony assay instead of recombinant growth factors. After 10 minutes at
4°C to solidify the medium, dishes were placed in humidified chambers at 37°C/5% CO2 for 14 to 21 days, after which
colonies containing greater than 20 cells were counted. Both the
concentrations of toxin reducing colony formation by 50%
(IC50) and the maximal log cell kill compared with controls
were calculated as previously described.15
In one experiment, cells from 4 AML samples treated with DT388-GMCSF
were assayed for colony formation in parallel in both the agarose-based
assay described above and in methylcellulose medium (StemCell
Technologies) with 30% FCS and 3 U/mL human erythropoietin (Epo;
StemCell), 10 ng/mL GM-CSF (Sandoz International, Basel, Switzerland),
10 ng/mL IL-3 (Sandoz), 50 ng/mL Steel factor (SF; Terry Fox
Laboratories, Vancouver, British Columbia, Canada), and 50 ng/mL flt-3
ligand (FL; Immunex) with equivalent results. All subsequent assays
were performed using the agarose-based assay and form the basis of the
experiments reported here.
Assays for normal bone marrow CFC.
After 4 to 48 hours of incubation in suspension culture, cells to be
assayed for burst-forming units-erythroid (BFU-E), colony-forming units-erythroid (CFU-E), CFU-granulocyte-macrophage (CFU-GM), and
CFU-granulocyte/macrophage/erythroid/megakaryocyte (CFU-GEMM) were
plated in methylcellulose-containing medium supplemented with 30% FCS
and 3 U/mL Epo (StemCell) to which was added 50 ng/mL SF and 20 ng/mL
each of human IL-6 (Terry Fox Laboratories), IL-3, GM-CSF, and G-CSF
(Amgen). After 18 to 21 days at 37°C, erythroid, GM, and
multilineage colonies were scored in situ. CFU-megakaryocyte (CFU-Mk)
were detected as previously described in a serum-free assay modified to
use a collagen base rather than agarose, to which was added 10 ng/mL
IL-3, 10 ng/mL IL-6, and 50 ng/mL thrombopoietin (Zymogenetics Inc,
Seattle, WA).24 After 18 to 21 days of incubation, Mk-containing colonies were identified in situ using immunocytochemical staining with an anti-CD41 monoclonal antibody (provided by P. Lansdorp, Terry Fox Laboratories) followed by alkaline
phosphatase/anti-alkaline phosphatase detection.24
| |
RESULTS |
Clinical history of AML patients.
Fifty previously untreated AML patients were studied. The age, type of
leukemia, and responsiveness of the patient to chemotherapy after
collection of the sample are shown in Table
1. There were 2, 7, 13, 11, 11, and 1 patient with the subtypes M0, M1,
M2, M4, M5, and M7, respectively, whereas for 5 patients the FAB type was not specified. No M3 or M6 subtypes were represented. A large proportion (78%) had presenting white blood cell counts
in excess of 50 × 109/L. The median age was 48.5 years (range, 9 to 82 years). Three non-AML samples (patients no. 51 and 52 with acute lymphoblastic leukemia [ALL] and patient no. 53 with prolymphocytic leukemia [PLL]) were also tested. Of the 47 patients who received remission induction chemotherapy, 22 (47%)
achieved CR.
GM-CSF receptor density.
Because the GM-CSF receptor population includes both high-affinity  ,
 chain complexes and low-affinity receptors consisting of the subunit only, we analyzed the presence of both types of receptor
complex on leukemic blasts. An example of one of the Scatchard plots is
shown in Fig 1. The results for all 53 leukemic patients are shown in Table 2. The
PLL sample and 1 ALL sample (no. 52) had no detectable high-affinity
receptors. The other ALL sample (no. 51) showed 93 high-affinity
receptors/cell, with a kd of 4.5 × 1011 mol/L and 216 low-affinity receptors, with a kd
of 5 × 109 mol/L. Eighty-eight
percent of AML patients (44/50) had 35 high-affinity receptors/cell,
and 74% (37/50) had greater than 100 high-affinity receptors/cell.
Among all 50 AML patients, for the high-affinity receptor, the mean ± SEM dissociation constant (kd) was 5 ± 1.3 × 1011 mol/L, and the median kd was 2 × 1011 mol/L. The mean and median kd of the
low-affinity receptor were both 1 ± 0.2 × 109 mol/L. Nonlinear regression analysis yielded
similar values (within 30%) for receptor numbers and kd on samples
from 4 normal marrow donors.
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| Fig 1.
Scatchard plot of the blasts of patient no. 1 with 3.36 × 106 cells per aliquot and 125I-GM-CSF
specific activity of 84 µCi/µg. The lower 6 concentrations and the
higher 4 concentrations were analyzed separately and the r2
values on both are .98.
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Colony formation by fresh AML blasts.
As shown in Table 2, 30 of 50 (60%) patient samples formed discrete
colonies of greater than 20 cells after 14 to 21 days in semisolid
medium. The plating efficiency of the various leukemic samples varied
by almost 3 orders of magnitude (from 8 to 5,950 CFU-L per
105 cells plated).
Inhibition of blast colony formation by DT388-GMCSF.
DT388-GMCSF reduced blast colony formation by at least 50% from 27 of
30 (90%, with 95% confidence interval 78%-98%) AML patient samples
(Table 2 and Fig 2). It had no effect on
colony formation from 2 AML samples (no. 46 and 47) that had less than
35 GM-CSF high-affinity receptors/cell. In contrast, the fusion toxin
inhibited colony growth by at least 50% from all but 1 AML sample with
35 high-affinity receptors/cell (no. 25). The 3 non-AML patients' blasts were either insensitive (no. 53 and 52) or showed minimal sensitivity to the toxin (no. 51). The median IC50 for
CFU-L among the 30 AML samples that formed colonies was 4 × 1012 mol/L (range, 5 to >4,000 × 1012 mol/L), whereas the corresponding values for
log kill of CFU-L in the same samples was 1.6 (range, 0 to 3.7; Table
2). In logistic regression analyses based on the 25 AML patients who
received remission induction chemotherapy, who had 35 high-affinity
receptors per cell, and whose samples grew colonies, there were no
significant associations between inhibition of colony growth (log cell
kill) and probability of complete response (two-tailed P = .36) or of resistant disease (P = .34). Similarly, there was
no significant association of the IC50 with CR (P = .33) or
resistant disease (P = .42).
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| Fig 2.
Colony growth inhibition after exposure of cells to
DT388-GMCSF in liquid culture for 48 hours followed by their plating in semisolid medium at 17 days (patient no. 34).
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Sensitivity of normal CFC to DT388-GMCSF.
Light-density clonogenic cells from 4 normal bone marrow samples were
also tested for sensitivity to the fusion toxin. As shown in
Table 3, an average of 56% of total normal
CFC survived (range, 33% to 102%), regardless of their lineage, after
being incubated for 48 hours with the maximum concentration of
DT388-GMCSF tested against both normal and malignant cells (4 × 109 mol/L). Failure of the fusion toxin to kill
normal CFC was not attributable to competition from endogenous GM-CSF
release during the 48-hour incubation period, because coincubated HL60
cells showed the same percentage of kill as in the absence of marrow cells (data not shown).
| |
DISCUSSION |
Recent clinical trials continue to show that the majority of AML
patients develop disease resistant to standard cytotoxic chemotherapy
drugs.25,26 Because DT388-GMCSF works by a unique mechanism
(inhibition of protein synthesis), its efficacy should not be affected
by most multidrug resistance phenotypes,15,17 and its in
vivo toxicity profile may be different than current cytotoxic
chemoradiotherapy regimens.27 Thus, such a reagent could be
useful in the treatment of both newly diagnosed and relapsed/refractory patients.
Before further clinical development, we sought experimental evidence
that AML blast progenitors from newly diagnosed patients could be
killed by the fusion toxin. Although we intended to test toxin
sensitivity in a series of different AML samples representative of the
entire spectrum of phenotypes in this disease, the need for relatively
large numbers of leukemic blasts to perform these experiments led to a
selection bias for patients presenting with high circulating blasts
counts. A large proportion of these patients (15/47 or 32%) failed to
respond to standard remission induction chemotherapy, consistent with
results from clinical trials showing that such patients have a poor
prognosis.25,26
Almost all (47/50) of the AML patients in this study had GM-CSF
receptors on their blasts. Our rate of receptor frequency paralleled
those reported by others.28,29 Kelleher et al30 reported a slightly lower receptor frequency on a small group of
patients using a cold saturation assay and two different types of
recombinant GM-CSF. Our higher receptor frequency may reflect our use
of the more sensitive hot saturation assay and/or the myelomonocytic differentiation of AML blasts from the M4 or M5 FAB
subgroups, which made up half of the samples we tested.
Although it is well known that clonogenic progenitors exist among the
malignant cells in patients with AML,31 their frequency and
the size and morphology of the colonies they produce are highly variable (Table 2).32 To demonstrate the sensitivity of
CFU-L to DT388-GMCSF quantitatively, it was necessary that the
malignant blasts from a given sample form a sufficient number of
discrete colonies in the semisolid assay used. Thirty of 50 (60%) AML
samples analyzed here met this criterion. Conclusions regarding the
sensitivity of CFU-L to the fusion toxin are thus restricted to this
group. Similarly, the maximum log kill detected in these assays was, in
many cases, determined by the clonogenicity of the untreated AML sample
(ie, even if 100% of CFU-L were killed by the toxin at low
concentrations, if only 10 colonies formed in the control assay the
maximum log kill detectable is 1). Patients whose leukemic blasts
contain a higher proportion of CFU-L have been reported to have a
poorer prognosis than AML patients in general.33
Nevertheless, even in this subgroup of patients, sensitivity of
clonogenic progenitors to DT388-GMCSF was observed here, with at least
50% inhibition of colony growth in 90% of patients.
The presence of GM-CSF receptors on CFU-L against which the toxin
showed cytotoxicity would be expected, because the drug must first bind
to target cells and internalize before intoxication. Both subunits of
the receptor must be present for ligand internalization.34 In fact, AML blasts from all the samples against which DT388-GMCSF had
activity were shown to have at least 35 high-affinity receptors per
cell. We did not find a significant correlation between either the
number of high-affinity receptors or their kd and their IC50 for the
drug. With this study's limited sample size, there may have been
insufficient statistical power to detect such associations. Another
possible explanation for this fact is that the receptor studies were
performed on the entire blast population rather than on the small
subset of clonogenic AML cells against which the toxicity of the drug
was tested. These latter cells may differ from the population as a
whole in their expression of GM-CSF receptors. Cells from the patient
with PLL and 1 ALL patient were both receptor negative and insensitive
to DT388-GMCSF, whereas the ALL patient sample that showed intermediate
numbers of high-affinity receptors had modest sensitivity
(IC50 6 × 1010 mol/L) to the drug.
The blasts of the 2 AML patients (no. 46 and 47), with 6 and 33 high-affinity receptors/cell, were insensitive to DT388-GMCSF. Although
these observations on lymphoid leukemias and AMLs with low numbers of
GM-CSF receptors on circulating blasts should be confirmed with larger
numbers, at present it appears appropriate to restrict future clinical
development of this reagent to AML patients with GM-CSF
receptor-positive blasts (35 receptors/cell).
CFU-L from the eight patients with refractory AML and blasts bearing 35 GM-CSF receptors per cell were assayed for DT388 GM-CSF sensitivity
in this study. All eight showed significant ( 1 order of magnitude)
inhibition of colony growth with low values of IC50 (eg, < 2 × 10-11 mol/L) for seven of the eight. These results extend
an earlier report on nine refractory patients by Parentesis et
al21 in which seven had DT-GMCSF-sensitive
blasts21 and are consistent with our results on
drug-resistant AML cell lines.15,35 We hypothesize that
targeted toxins are, in general, less affected by multidrug resistant
phenotypes induced by conventional cytotoxic drugs or radiotherapy.
Confirmation of this hypothesis will require testing of a larger group
of relapsed/refractory patients' blasts.
In contrast to the marked toxicity of DT388-GMCSF on AML progenitors,
its effect on most normal bone marrow clonogenic cells was
insignificant, even after exposure to the highest concentrations of
toxin tested for 48 hours. These results (Table 3) are consistent with
those reported by others.17,19,20 Further evidence for the
lack of effect of this reagent on more primitive normal hematopoietic precursors that form cobblestone areas in long-term culture has also
recently been published.36 Taken together, these findings support the further preclinical development of DT388-GMCSF as a novel
treatment agent for AML patients with both chemotherapy-sensitive and
drug-resistant disease.
| |
FOOTNOTES |
Submitted November 24, 1997;
accepted March 10, 1998.
Supported by LSA Grant No. 6114-98 (A.E.F.) and by the National Cancer
Institute of Canada (D.E.H. and C.J.E.) with funds from the Terry Fox
Run. C.J.E. is a Terry Fox Cancer Research Scientist of the National
Cancer Institute of Canada. Also supported by NIH Grant No. CA76178
(A.E.F.), Grant No. CA 12213 (C.L.W.), and the SWOG Statistical Center
(K.J.K.).
Address reprint requests to Arthur E. Frankel, MD, Hanes 4046, Med
Center Drive, Winston Salem, NC 27157.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract