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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 1981-1988
An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor
Stimulates Megakaryocytopoiesis
By
Bijia Deng,
Naheed Banu,
Beth Malloy,
Philip Hass,
Jian Feng Wang,
Lisa Cavacini,
Dan Eaton, and
Hava Avraham
From the Divisions of Experimental Medicine and Hematology/Oncology,
Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine,
Boston, MA and Genentech Inc, South San Francisco, CA.
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ABSTRACT |
Thrombopoietin (TPO) is a hematopoietic growth factor that
stimulates megakaryocytopoiesis and platelet production in vivo and
promotes the development of identifiable megakaryocytes in vitro. We
have developed a murine monoclonal antibody, BAH-1, raised against
human megakaryocytic cells, which specifically recognizes the c-Mpl
receptor and shows agonist activity by stimulating megakaryocytopoiesis
in vitro. BAH-1 antibody specifically binds to platelets and to
recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone
supported the formation of colony-forming unit-megakaryocyte
(CFU-MK) colonies. The combination of BAH-1 plus
interleukin-3 or of BAH-1 plus human TPO significantly increased the
number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody
stimulated the proliferation and maturation of primary bone marrow
megakaryocytes in a dynamic heterogeneous liquid culture system.
Individual large megakaryocytes as well as small megakaryocytic cells
were observed in cultures of CD34+ CD41+
cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature
megakaryocytes as observed by an increase in the detectable numbers of
acetylcholinesterase-positive megakaryocytes. No effects of BAH-1
antibody were observed on colony-forming unit-granulocyte-macrophage,
burst-forming unit-erythroid, or colony-forming unit-erythroid
colonies. In vivo studies showed that BAH-1, alone or in combination
with TPO, expands the numbers of megakaryocytic progenitor cells in
myelosuppressed mice. This antibody should prove useful in
understanding the structure-function aspects of the c-Mpl receptor as
well as in evaluating the effects of the sustained activation of this
receptor in preclinical models of severe thrombocytopenia.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THROMBOPOIETIN (TPO) is the primary
regulator of physiological platelet production. TPO has dramatic
effects on both the proliferation and differentiation of megakaryocytes
in vitro and in vivo and is the most potent thrombopoietic agent
described to date.1-13 At therapeutic doses, TPO causes as
much as a 10-fold increase in circulating platelet
levels.12 The central role of TPO in megakaryocytopoiesis
and thrombopoiesis is further shown by the severe thrombocytopenic
phenotype of mice rendered null for the expression of either TPO or its
receptor c-Mpl.14-16 The similarity in the phenotype of the
TPO and c-Mpl knockout mice shows that the system is nonredundant and
that there is one receptor for TPO and one ligand for c-Mpl. Because of
this, it is presumed that binding of TPO to c-Mpl is solely responsible
for its activation.
The cell surface receptor (c-Mpl) for TPO is a member of the
hematopoietic growth factor receptor superfamily.17
Extracellular domains of members of this family are typically composed
of multiple  -sandwich modules related to the fibronectin type-III
immunoglobulin fold, with a characteristic ligand-binding domain formed
from two adjacent -sandwich structures.18 The mechanism
by which TPO activates c-Mpl appears to be similar to that of other
hematopoietic growth factors (such as erythropoietin [EPO], growth
hormone [GH], prolactin [PRL], and granulocyte
colony-stimulating factor [G-CSF]), that are triggered by
ligand-induced receptor homodimerization.19-21 Recently,
two families of small peptides that bind to the c-Mpl and compete with
the binding of the natural ligand TPO were identified from recombinant
peptide libraries.22 The peptide dimer stimulated the in
vitro proliferation and maturation of megakaryocytes from human bone
marrow cells.22
An antibody having agonist activity that stimulates c-Mpl might serve
as an attractive therapeutic option in situations in which a prolonged
half-life is needed and in which less frequent administration is
desired. Furthermore, mapping the binding site on the c-Mpl receptor of
an agonist monoclonal antibody (MoAb) would improve our understanding
of the structure-function relationships of this receptor. To these
ends, we report on the development of a murine MoAb, termed BAH-1,
raised against human megakaryocytic cells that specifically recognizes
the c-Mpl receptor, shows agonist activity by stimulating
megakaryocytopoiesis in vitro, and also expands the numbers of
megakaryocytic progenitor cells in myelosuppressed mice.
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MATERIALS AND METHODS |
Immunization and cell fusion.
BALB/C mice (Jackson Laboratories, Bar Harbor, ME) received repeated
injections of 107 CMK cells emulsified in complete Freunds
adjuvant according to a previously reported protocol.23-27
A final booster of 2 × 105 human primary bone marrow
megakaryocytes plus 106 CMK cells was injected 3 to 4 days
before the animals were sacrificed.
Spleen cells from the immunized mice were fused with a mouse myeloma
cell line (X653). These spleen cells (approximately 1 × 108) were fused with 2 × 107 myeloma
cells by the addition of 1 mL of 40% polyethylene glycol (Sigma
Chemical Corp, St Louis, MO). They were then diluted with 15 mL of
Dulbecco's modified Eagle's medium (DMEM), centrifuged, and rediluted
into a complete (10% fetal calf serum) selective medium containing
hypoxanthine/aminopterin/thymidine at 2 × 106
cells/mL. The cells were then distributed into 96 wells (100 µL) in
hypoxanthine/aminopterin/thymidine medium.24 After 10 to 16 days, duplicate aliquots of the supernatants were assayed for cell
enzyme-linked immunosorbent assay (ELISA) binding. Hybridoma cells from
positive testing wells were then transferred to 24 wells containing 0.5 mL of hypoxanthine and thymidine medium. After the cells were grown and
retested, they were cloned and recloned by limiting dilution into
96-well plates. For the antibody assays, the supernatants from the
hybridoma cells were tested directly.
A screening procedure with a whole cell ELISA technique was used to
detect antibodies that specifically recognized the human megakaryocytic
cell lines CMK, DAMI, Mo7e, and CMS but not T cells, B cells, and
monocyte-macrophages.28,29 After the identification of
candidate antimegakaryocyte antibodies, screening for recognition of
known megakaryocytic surface structures was done by cross-blocking studies with a panel of antibodies against the integrins GpIb and
GpIIb/IIIa.28,29 One MoAb (BAH-1) that appeared to be
specific for megakaryocytes and did not recognize GpIb or GpIIb/IIIa
was further characterized. The BAH-1 MoAb was isotyped by using a MoAb
isotyping kit (Innogenetics, Antwerp, Belgium), and found to be IgG1
kappa. BAH-1 hybridoma cells were injected intraperitoneally into
BALB/C mice primed with pristine, and the antibody-containing ascites
fluid was collected 2 to 3 weeks later and affinity-purified as
described.28
Growth factors.
Recombinant human interleukin-3 (IL-3) or recombinant murine IL-3,
human granulocyte-macrophage colony-stimulating factor (GM-CSF) and
human IL-6 were obtained from R&D Systems (Minneapolis, MN). These
cytokines were determined to be free of endotoxin contamination. Plateau doses of each factor were determined from dose-response curves
for each assay. Recombinant human thrombopoietin (hTPO) and murine
thrombopoietin (mTPO; both from Genentech Inc, South San Francisco, CA)
were used at 100 ng/mL as determined from dose-response curves in the
megakaryocyte progenitor assays (colony formation and liquid cultures).
In some experiments, as indicated, we used various dilutions of TPO or
IL-3 (10-100 ng/mL) to assess the synergistic effects of both cytokines
under conditions of subconcentration or optimum concentration on the
megakaryocytic lineage.
Marrow megakaryocytes.
Human bone marrow was obtained by aspiration from the iliac crests of
normal donors who gave their informed consent in a protocol approved by
the Deaconess Hospital Institutional Review Board. The marrow was
aspirated into preservative-free heparin (Sigma) and separated by
centrifugation through Ficoll-Hypaque (Pharmacia Biotech Inc,
Piscataway, NJ) at 1,200g at room temperature for 30 minutes.
After two washes with sterile phosphate-buffered saline (PBS), the
cells were resuspended in Iscove's modified Dulbecco's medium with
20% fetal calf serum (FCS), penicillin/streptomycin, and L-glutamine;
seeded onto T-75 tissue culture flasks (Corning, Corning, NY); and
incubated at 37°C in 5% CO2. After 24 hours, the
nonadherent cells were gently removed. Human marrow megakaryocytes were
isolated by a method using immunomagnetic beads with antihuman GpIIb/IIIa MoAb as described previously.11 The cells that
rosetted with the immunomagnetic beads were collected with a dynal
magnetic particle concentrator (Dynabeads M-450; Dynal Inc, Great Neck, NY) and were washed three times with a megakaryocyte medium, which consisted of Ca2+  Mg2+ free PBS
containing 13.6 mmol/L-sodium citrate, 1 mmol/L theophylline, 1%
bovine serum albumin (BSA), fraction V (Sigma), and 11 mmol/L glucose,
adjusted to pH 7.3 and an osmolarity of 290 mOSM/L. After purification,
cells were labeled using a MoAb against von Willebrand's factor (MoAb
4F9; AMAC Inc, Westbrook, ME), and more than 95% of the cells were
stained. Twenty milliliters of bone marrow aspirate generally yielded
about 1 × 105 megakaryocytes. Contaminating cells
(1%-5%) were essentially monocytes and macrophages. Cells were
cultured in RPMI-1640 supplemented with 2% platelet poor plasma
(PPP)11 at 37°C in a 5% CO2 fully humidified atmosphere for 24 hours. Monocytes and macrophages were
identified by morphology after May-Grunwald-Giemsa staining and by
positive antibody staining using a MoAb directed against CD14
(monocytes), CD15 (granulocytes), CD16 (IgG Fc receptor-natural killer
cells, granulocytes, and macrophages; AMAC Inc). These analyses
indicated that the maximum potential degree of contamination of bone
marrow megakaryocytes after 24 hours was about 5% to 10%.
Isolation of CD34+ cells by the immunomagnetic bead
technique.
CD34+ cells were isolated as described.12 Cells
were first incubated at 4°C for 30 minutes with the
CD34+ MoAb at a concentration of 10 mg/mL, and then with
paramagnetic beads coupled with goat antibody to mouse IgG (Dynabeads
M-450; Dynal) with a bead-to-target cell ratio of 5:1.
CD34+ cells were isolated by magnetic separation and
detached from the beads by chymopapain treatment (Sigma; 130 U/mL for
10 minutes), which allows for the collection of CD34+ cells
free of beads.
Megakaryocyte progenitor assay.
Low-density bone marrow cells were cultured in a semisolid medium by
using the plasma clot technique.11 The medium consisted of
RPMI-1640, 1% deionized BSA, 20 mg/mL asparagine, 28 mg/mL CaCl2, 10% PPP, and 2.5 × 105
nonadherent bone marrow cells in the absence or presence of various dilutions of the MoAb BAH-1. Ten percent citrated bovine plasma (GIBCO,
Gaithersburg, MD) was added as the last product. The PPP and citrated
bovine plasma used in these cultures were assayed and determined to be
devoid of any detectable IL-6 or endogenous transforming growth
factor- (TGF-) by specific immunoenzymatic assays for IL-6 and
TGF-, respectively (R & D Systems). Cultures were incubated for 12 days at 37°C in duplicate. Quantitation of colonies was performed
by an ABC labeling kit (Vector Laboratories, Burlingame, CA) by using
anti-GpIIIa antibodies. Each dish was entirely scanned under a
microscope at day 12 of culture, and each cluster of three or more
megakaryocytes was scored as a colony. For the limiting dilution
experiments, CD34+ CD41+ cells were directly
sorted into 96-well tissue culture plates and the numbers of
megakaryocytes were identified by a GpIIb/IIIa cell-based
ELISA.11
Human megakaryocytic cell lines.
The megakaryoblastic cell lines CMK,30,31 DAMI, and Mo7e
were provided by Dr T. Sato (Chiba University, Chiba, Japan), Dr S. Greenberg (Brigham and Women's Hospital, Boston, MA) and Dr J. Hoxie
(University of Pennsylvania, Philadelphia, PA), respectively. Each cell
line was cultured as previously described.32 Jurkat T cells
were obtained from the American Type Tissue Collection and maintained
in liquid culture according to the specifications in the catalog.
Murine megakaryocyte assay.
To assess megakaryocytic differentiating activity, a single
megakaryocyte growth assay was used.31 Single cell
populations from bone marrow were prepared from the femurs of normal
C57/BL6 mice. This preparation was performed by flushing the bones with DMEM containing 10% FCS. Immature megakaryocyte populations were obtained in 1.07 to 1.085 g/cm 3 fractions, from a
suspension of single bone marrow cells separated in a Percoll gradient.
The fractionated cells were cultured in 10% FCS in DMEM for 5 days at
37°C in a 10% CO2 humidified incubator. This procedure
was performed in the presence of titrated doses of growth factors.
Cultures were dried and stained for acetylcholinesterase. Growth and
maturation of immature megakaryocytes were quantitated by assessing the
number of single large megakaryocytes detected by light microscopy.
Mice treatments and assays.
For the myelosuppression experiments, 6- to 9-week-old female BALB/C
mice received a single intraperitoneal injection of 1.2 mg carboplatin
and 350 cGy whole-body 137Cs irradiation (Gammacell 40 irradiator; Atomic Energy of Canada Radiochemical Co, Kanata, Canada)
on day 0. The following day, the mice were begun on daily
intraperitoneal injections of vehicle (20 mmol/L Tris, pH 8.1/0.9%
NaCl/0.25% rabbit serum albumin), recombinant murine TPO (40 kU/mouse/d) in vehicle, purified BAH-1 antibody (5 mg/mouse), or both
TPO (40 kU) and BAH-1 antibody (5 mg/mouse).
Animals were sacrificed 13 to 14 days after the initiation of
treatment, which was 2 to 3 days before the onset of platelet recovery
in the TPO-treated mice.
After sacrifice by cervical dislocation, the femurs of each study mouse
were harvested and single-cell suspensions were prepared by using
standard techniques.33,34 From 0.5 to 2 × 105 cells/mL were plated for megakaryocytic colony
formation (CFU-MK) using 20 ng/mL murine IL-3 plus 7 ng/mL murine TPO
in agar as previously described.8 As these cultures
contained optimal levels of IL-3, granulocyte-macrophage colonies
(CFU-GM) were also enumerated. Assays for erythroid bursts (BFU-E) were
performed using recombinant human EPO as previously
described.8 Late erythroid progenitors (CFU-E) were assayed
in a plasma clot in the presence of 0.5 U/mL EPO.35 Each
assay was performed in duplicate.
Flow cytometric analysis of surface protein expression.
To detect the potential surface binding proteins that bind BAH-1, we
used flow cytometric analysis (FACS staining). Cells were washed with
sterile PBS, and 1 × 106 cells were resuspended in
0.1 mL of PBS. Cells were incubated with 10 mL of the BAH-1 MoAb or
GpIIIa antibodies, mouse IgG as a control (Immunotech Inc, Westbrook,
ME) or PBS at 4°C for 20 minutes. Fluorescein isothiocyanate
(FITC)-conjugated goat antimouse IgG or goat antirabbit IgG was added
at a final dilution of 1:500 and followed by incubation for 20 minutes
at 4°C. Cells were washed twice and resuspended in 0.5 mL of 1%
(vol/vol) paraformaldehyde in PBS. Cells were then analyzed by flow
cytometry.
Immunoprecipitation and Western immunoblotting.
CMK cells (2 × 106/mL) were serum-starved
for 4 to 5 hours in RPMI-1640 medium. Cells were centrifuged, then
resuspended at 107 cells/mL in the RPMI-1640. Cells (20 × 106/precipitation) were placed on ice and lysed by
the addition of 1/3 volumes of 3× lysis buffer (40 mmol/L
Tris-HCl, pH 7.4/2 mmol/L MgCl2/2 mmol/L
CaCl2/20% glycerol/2% NP-40/2 mmol/L
Na3VO4/20 mg/mL leupeptin/20 mg/mL aprotinin/4
mmol/L phenylmethyl sulfonyl fluoride [PMSF]). Lysates were
centrifuged at 10,000g for 15 minutes. The MoAbs BAH-1 or Mpl-R
(Genzyme, Cambridge, MA) were added to the supernatant at 5 mg/precipitation. Tubes were incubated by rocking at 5°C for 3 hours, and then 40 mL of 1:1 Protein G-Sepharose (Pierce, Rockford, IL)
were added. After 1 and a half hours, lysates were washed three times
with 1× lysis buffer. Sodium dodecyl sulfate (SDS) sample buffer
was added to the washed beads and samples were run on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5%
acrylamide). SDS polyacrylamide gels were electrophoretically transferred onto nitrocellulose membranes (BioRad, Hercules, CA). The
membranes were blocked with 4% BSA in PBS/0.1% Tween 20 (PBST) and
then incubated with the BAH-1 MoAb (0.2 mg/mL) for 1 and a half hours
or with Mpl-R MoAb (0.2 mg/mL). Membranes were then washed three times
in PBST and incubated for 45 minutes in horseradish peroxidase-linked
secondary antibody (Amersham Corp, Arlington Heights, IL) diluted in
PBST. Transfers were washed three times in PBST and developed by the
enhanced chemiluminescence (ECL) method (Amersham).
Binding of BAH-1 to platelets and c-Mpl.
Platelet rich plasma was prepared by centrifugation of citrated whole
blood at 400g for 5 minutes. Binding studies were conducted within 3 hours of collection. 125I-BAH-1 was prepared by
indirect iodination and yielded a specific activity of 15 to 50 mCi/mg
protein. 125I-BAH-1 (100 pmol/L) was incubated with 4 × 107 platelets in plasma at 37°C for 30 minutes
with varying concentrations of unlabeled BAH-1 in triplicate. The
reaction mixture was overlayed on 1 mL of Hank's balanced salt
solution containing 0.5% BSA and 20% sucrose, then microcentrifuged
at 13,500 rpm for 5 minutes. The supernatants were aspirated, tube
bottoms containing the cell pellets were cut off, and
platelet-associated radioactivity was determined.
The binding of BAH-1 to recombinant c-Mpl was determined by using a
solid phase assay as described.36 Ninety-six-well
immunoplates were coated with 50 mL of rabbit antihuman IgG Fc (Jackson
Labs; 2 mg/mL in carbonate buffer, pH 9.6) overnight at 4°C. After
blocking for 1 hour with PBS containing 0.5% BSA, the plates were
incubated with conditioned media from the Mpl-IgG transfected 293 cells. Plates were subsequently washed three times with PBS containing 0.5% BSA and 0.05% Tween 20 (assay buffer) and 125I-BAH-1
(100 pmol/L) was added with varying concentrations of unlabeled BAH-1.
After 1 hour, the plates were washed five times with assay buffer and
bound 125I-BAH-1 was eluted with 4% SDS in 0.1 mol/L
acetic acid and then quantitated in a gamma counter.
Statistical analysis.
The results were expressed as the mean ± standard error of the mean
(SEM) of data obtained from three or more experiments performed in
triplicate. Statistical significance was determined using the
Student's t-test.
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RESULTS |
Characterization of the MoAb BAH-1.
We derived one murine MoAb (BAH-1), which appeared to specifically
interact with the human megakaryocytic cell line DAMI. Immunofluorescence staining showed that more than 90% of the DAMI cells stained positive with BAH-1 MoAb. Similar results were obtained with other megakaryoblastic cell lines, such as CMK, CMS, and Mo7e
(data not shown). No staining was observed when Jurkat T cells were
used (Fig 1). In addition, this antibody
did not recognize the major megakaryocytic glycoproteins Ib or IIb/IIIa
(data not shown). Immunoprecipitation and Western blot analysis showed
that this MoAb reacted specifically with the c-Mpl protein
(Fig 2). BAH-1 immunoprecipitated an 84-kD
protein from CMK cell lysates that cross-reacted with a known c-Mpl
antibody (Mpl-R).
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| Fig 1.
Flow cytometric analysis of BAH-1 surface protein
expression in a DAMI megakaryocytic cell line. Antigen expression was
evaluated by FACS staining using BAH-1 and Mpl-R MoAbs (commercially
available from Genzyme, MA), CD61 or control MoAb on DAMI (A) and
Jurkat T cells (B). X653, supernatant from the mouse myeloma cell line
used in cell fusion; 12R, a control nonrelevant MoAb (1:1,000); CD61, a
positive control for megakaryocytic cells; BAH-1 Asc, ascites of BAH-1
(1:1,000 dilution); BAH-1 Sup, BAH-1 supernatant (1:1,000 dilution).
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| Fig 2.
Immunoprecipitation of Mpl-R protein. CMK cell extracts
were immunoprecipitated with BAH-1 antibodies and subjected to SDS-PAGE
followed by Western blotting using control MoAb or BAH-1 antibodies (A)
or Mpl-R antibodies (B) as described in the Materials and Methods
section. Arrow indicates the position of the c-Mpl. The reactive
proteins were detected by using the ECL system (Amersham).
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We also determined the ability of BAH-1 to bind to platelets and
recombinant c-Mpl (Fig 3). In
equilibrium binding studies, a kd of 2.3 nmol/L was determined for
BAH-1 binding to platelets. This compares to a kd of 200 pmol/L for
TPO.36 By using a recombinant truncated version of c-Mpl,
which consists of the extracellular domain of c-Mpl fused to an Fc
domain of IgG,36 we found that BAH-1 also bound
specifically to purified c-Mpl with a kd of 0.61 nmol/L. These results
show that BAH-1 specifically binds c-Mpl with high affinity.
Interestingly, competition experiments showed that BAH-1 did not
inhibit TPO binding to c-Mpl, suggesting that TPO and BAH-1 bind to
different sites on the c-Mpl receptor (data not shown).
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| Fig 3.
Binding of BAH-1 to human platelets and recombinant
c-Mpl. Equilibrium binding studies for 125I-BAH-1 were
performed on platelets (A) and c-Mpl-IgG fusion protein (B) as
described in the Materials and Methods. The kd was determined as
described.40
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Functional characterization of BAH-1 MoAb.
The effects of BAH-1 MoAb on megakaryocytopoiesis were evaluated in
liquid suspension and fibrin clot megakaryocytopoiesis assays. For the
liquid suspension assay, purified CD34+ cells (5 × 104/mL) were cultured in serum-free medium in the presence
of various concentrations of BAH-1 for 10 days. As shown in
Fig 4, BAH-1 stimulated the production of
megakaryocytes in these cultures. A plateau was reached at 100 ng/mL of
BAH-1, a level of stimulation that is similar to that caused by 100 ng/mL of TPO (Fig 4). Similarly, in the fibrin clot system BAH-1 also
stimulated the production of CFU-MK progenitors
(Fig 5A and B). In combination with IL-3, BAH-1 acted additively. No effects were observed on CFU-GM, BFU-E, or
CFU-E colonies when BAH-1 antibody was used in various concentrations.
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| Fig 4.
Effects of TPO, IL-3, and BAH-1 MoAb on megakaryocyte
differentiation of CD34+ cells. CD34+ cells
(1 × 104/mL) were cultured in 500 mL of serum-free
culture medium. After 10 days, the increase in megakaryocytes was
determined by ELISA using anti-GpIIb/IIIa antibodies. Results represent
the mean optical density (OD) ± SEM of three independent experiments.
*Statistically significant compared with PBS (P < .05).
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| Fig 5.
Effect of BAH-1 MoAb on human CFU-MK colonies. (A)
CD34+ bone marrow cells were plated at 5 × 103/mL in the fibrin clot system (see the Materials and
Methods). Results are expressed as the means ± SEM of megakaryocyte
colonies. Experiments were performed in triplicate in four
assays. *Statistically significant compared with PBS (P < .05). (B) Photographs of CFU-MK colonies in the presence of BAH-1
(B-I) or TPO (B-II).
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We also determined the ability of BAH-1 to directly stimulate the
proliferation of early megakaryocyte progenitors. In limiting dilution
experiments, CD34+ CD41+ cells were plated at a
concentration of 1 to 50 cells (100 mL volume) in the presence of TPO,
IL-3, or BAH-1 alone or in combination. After 5 days in culture,
overall expansion was determined and megakaryocytes quantitated by
staining with an anti-IIb/IIIa antibody. As shown in
Fig 6, the effect of BAH-1 was similar to
that of TPO. The percentage of positive megakaryocytes in each well was determined to be approximately 40% to 70%. Individual large
megakaryocytes as well as small megakaryocytic cells were observed.
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| Fig 6.
Effect of BAH-1 on CD34+
CD41+ cells. CD34+ CD41+
cells were cultured under serum-free conditions in the presence of
rhTPO, IL-3 (100 ng/mL), the optimal plateau concentration as
determined for IL-3 with this assay, or various concentrations of
BAH-1. On day 5, megakaryocytes in culture were quantitated visually by
using an inverted microscope. *Statistically significant compared with
PBS (P < .05).
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Effect of BAH-1 on murine megakaryocytopoiesis.
Dot blot analysis indicated that BAH-1 also cross-reacts with murine
Mpl (data not shown). Because of this, we determined whether BAH-1 was
also an agonist for murine megakaryocytopoiesis. In a liquid suspension
assay,31 BAH-1 stimulated the proliferation of immature
megakaryocytes (Fig 7). However, BAH-1
failed to stimulate murine CFU-MK colony formation
(Fig 8) although it did appear to synergize
with TPO or IL-3 in stimulating murine CFU-MK formation (Fig 8). These
results suggest that BAH-1 does not induce complete differentiation of
murine megakaryocytes.
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| Fig 7.
Effect of BAH-1 MoAb on murine immature megakaryocytes by
using a single megakaryocyte growth assay. Single megakaryocytes were
scored as the number of acetylcholinesterase-positive cells per
fractionated 5 × 104 bone marrow cell cultures. Results
are the means ± SEM from triplicate cultures from three experiments.
*Results were significantly different (P < .01) from FCS
control.
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| Fig 8.
Effect of BAH-1 MoAb on murine CFU-MK colonies. CFU-MK
megakaryocytes were scored as the number of colonies with three cells
or more per 105 unfractionated cells. BAH-1 (1 µg/mL) was
used in various dilutions as indicated. Results are the means ± SEM
from triplicate cultures from three experiments. *Results were
significantly different (P < .01) from FCS control.
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Effect of BAH-1 on hematopoiesis in myelosuppressed mice.
The effects of BAH-1 alone or in combination with TPO on hematopoietic
progenitor cell numbers during the recovery phase after myelosuppressive therapy were evaluated in myelosuppressed mice. Only a
modest increase in the numbers of CFU-MK colonies was observed in the
marrow of BAH-1-treated mice (Table 1),
whereas an increase in the numbers of CFU-MK colonies was observed in
the BAH-1 plus TPO-treated mice (Table 1). No effects of BAH-1 were
observed on the CFU-GM, BFU-E, or CFU-E colonies (Table 1). The modest effect of BAH-1 in this model is likely because of the relatively modest effect of BAH-1 on murine megakaryocytopoiesis.
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Table 1.
Marrow Hematopoietic Progenitor Cell Levels After
the Administration of BAH-1, TPO, Control MoAb, or TPO plus BAH-1
to Myelosuppressed
Mice
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DISCUSSION |
We have generated a murine MoAb, BAH-1, against human megakaryocytic
cells that specifically recognizes the TPO receptor, c-Mpl. BAH-1
specifically bound to platelets and recombinant c-Mpl with high
affinity and had agonist activity in various assays of in vitro human
and murine megakaryocytopoiesis, including the generation of CFU-MK
megakaryocyte progenitors and the production of mature
GpIIb/IIIa-expressing megakaryocytes in liquid cultures of
heterogeneous bone marrow cells. Although BAH-1 was able to trigger
cell proliferation and differentiation of human megakaryocytic precursors and immature murine megakaryocytes, by itself it failed to
stimulate murine CFU-MK colony formation (Fig 7). However, it did
synergize with IL-3 and TPO to stimulate murine CFU-MK formation. From
these results, we conclude that BAH-1 is an agonist antibody specific
for c-Mpl and is capable of stimulating human megakaryocyte growth and
maturation.
TPO has an effect on stem cells as well as erythroid
progenitors.15,34 However, BAH-1 alone had no effects on
BFU-E and CFU-E colonies under the conditions tested, as
described.15 The lack of potency of BAH-1 on the erythroid
lineage could be caused by a cross-species effect, because BAH-1 is a
murine antibody being tested in a human system. Alternatively, a
cooperative effect with EPO could be needed to see an effect of BAH-1
on this lineage.
Homodimerization of the c-Mpl receptor by TPO on the cell surface is
believed to be the key event in TPO-induced signal
transduction.17 In support of this, TPO has recently been
shown to contain two receptor binding sites that function to dimerize
c-Mpl.37 Similarly, because of its bivalency, BAH-1 likely
acts as an agonist by inducing homodimerization of c-Mpl. Indeed,
monovalent Fab fragments of BAH-1 antibody, which cannot form receptor
dimers, did not stimulate megakaryocytopoiesis (data not shown). These
results suggest that the mechanism through which BAH-1 stimulates
megakaryocytopoiesis is by "self-antagonism," which has been
described for agonist antibodies for GH, PRL, and EPO
receptors.35,38,39
BAH-1 offers several opportunities for improving our understanding of
the biology of megakaryocytopoiesis as well as its potential therapeutic applications. More detailed mapping of the binding site of
this MoAb on c-Mpl may help define the sequences and confirmation of
the native receptor, which are necessary and sufficient for activation
and subsequent signal transduction. Studies of different c-Mpl
constructs are in progress to achieve this aim. It is interesting to
note that BAH-1 does not antagonize TPO binding to c-Mpl, suggesting that they have different binding sites.
Currently, it is not known if BAH-1 stimulates platelet production in
vivo. In a murine myelosuppressive model, BAH-1 only modestly affected
megakaryocytopoiesis. This may be caused by species specificity because
BAH-1 was much more effective in stimulating human versus murine
megakaryocytopoiesis in vitro.
Although the most immediate clinical trials are of recombinant human
TPO, there may be certain opportunities for the use of a humanized
agonist MoAb. Humanizing murine antibodies have successfully yielded a
number of products currently in clinical trials that have minimal
antigenicity while sustaining affinity and functional potency in the
recognition of human antigens. Because of the prolonged half-life of
antibodies, it might be feasible to administer an agonist antibody to
the c-Mpl receptor on an intermittent basis, thereby sustaining the
stimulation of megakaryocytopoiesis in patients with a compromised
production of cells of this lineage.
| |
FOOTNOTES |
Submitted January 5, 1998;
accepted May 11, 1998.
Supported in part by Genentech Inc and National Institutes of Health
Grant No. RO1 51456.
Address reprint requests to Hava Avraham, PhD, Division of Experimental
Medicine, Harvard Institutes of Medicine BIDMC, 4 Blackfan Circle, 3rd
Floor, Boston, MA 02115.
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.
| |
ACKNOWLEDGMENT |
We thank Dr Jerome E. Groopman for his helpful and valuable
discussions. We are grateful to Janet Delahanty for editing and preparation of the figures for this manuscript, as well as Evelyn Gould
for her help with the figures. We also thank Tee Trac for her typing
assistance.
| |
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Abstract
Introduction
Abstract