Stem Cells, Vol. 17, No. 4, 203-209,
July 1999
© 1999 AlphaMed Press
Immunogenicity of Thrombopoietin Mimetic Peptide GW395058 in BALB/c Mice and New Zealand White Rabbits: Evaluation of the Potential for Thrombopoietin Neutralizing Antibody Production in Man
Mark De Serres,
Byron Ellis,
John E. Dillberger,
Sharon K. Rudolph,
Jeff T. Hutchins,
Christine M. Boytos,
Debra L. Weigl,
Randolph B. DePrince
Glaxo Wellcome Inc., Research Triangle Park, North Carolina, USA
Key Words. Immunogenicity • Thrombopoietin • Mimetic peptide • GW395058 • Mice • Rabbits • Neutralizing
antibody
Mr. Mark de Serres, Glaxo Wellcome Inc., Department of International Development Support, Division of Bioanalysis and Drug Metabolism, 5 Moore Drive, Research Triangle Park, North Carolina 27709, USA.
 |
ABSTRACT
|
|---|
Administration of exogenous proteins and peptides as therapeutics
carries with it the potential for immune system recognition and the
development of neutralizing antibodies to endogenous regulatory
proteins. PEGylation of proteins typically reduces their
immunogenicity in vivo. GW395058 is a PEGylated peptide thrombopoietin
receptor (TPOr) agonist being evaluated for the treatment of
chemotherapy-induced thrombocytopenia. Although GW395058 shares no
homology with TPO, it does compete with TPO for binding to a common
receptor, and a similarity in local structure could result in shared
epitopes. Thus GW395058 could elicit TPO-neutralizing antibodies. In
this study, we evaluated the immunogenicity of GW395058 in mice, the
potential of rabbit antibodies elicited by immunizations with the
non-PEGylated parent peptide AF15705 to cross-react with recombinant
human (rHu) TPO, and the potential of mouse anti-rHuTPO antibodies
elicited by repeated dosing with rHuTPO to cross-react with
AF15705. GW395058-dosed mice failed to produce antibodies to AF15705
or rHuTPO. Mouse anti-rHuTPO did not cross-react with AF15705 and
rabbit anti-AF15705 antibodies failed to cross-react with
rHuTPO. GW395058 caused no immune-mediated lesions in mice, but rHuTPO
suppressed megakaryocytopoiesis and caused B-lymphocyte hyperplasia in
lymphoid tissues consistent with antigenic stimulation. These data
suggest that the potential for an immune response to GW395058 in man
would be low. Stem Cells
1999;17:203-209
|
Introduction
|
|---|
Neutropenia and thrombocytopenia are chemotherapy-induced side
effects associated with significant morbidity, including spontaneous
bleeding. Although recombinant interleukin 11 (IL-11) has been
approved for the treatment of chemotherapy-induced thrombocytopenia,
its use has been associated with notable side effects [1-3]. Standard practice is to manage
thrombocytopenia by administering platelet transfusions, which carry a
risk of secondary infection, as well as the eventual production of
neutralizing antibodies to platelets. Thrombocytopenia often limits
the maximum chemotherapy dose and the dosing regimen that can be
administered. Reducing the magnitude of thrombocytopenia might allow
administration of higher doses of chemotherapy or dose
intensification, which could result in improved disease control.
Recently, the lineage-specific cytokine that regulates platelet
production, thrombopoietin (TPO), was identified [4]. Two recombinant forms of human
TPO are under investigation: full-length glycosylated recombinant
human TPO (rHuTPO) [5]
and PEGylated megakaryocyte growth and development factor
(PEG-rHuMGDF). PEG-rHuMGDF is a truncated nonglycosylated form of
rHuTPO that has been derivitized with polyethylene glycol [6]. In mice, dogs, and primates,
rHuTPO and PEG-rHuMGDF promote platelet production and reduce
chemotherapy-induced thrombocytopenia [7-14]. In humans, rHuTPO and PEG-rHuMGDF promote
platelet production and reduce chemotherapy-induced thrombocytopenia
[15-19]. Unfortunately, evidence of
TPO-neutralizing antibodies in patients participating in cancer and in
platelet donor clinical trials forced the discontinuation of
PEG-rHuMGDF development [20]. This outcome may affect the future
clinical applications of rHuTPO.
Shortly after TPO was isolated, two families of small peptides
that bind to the human TPO receptor and compete with the binding of
TPO were identified from recombinant peptide libraries. Screening of
variant libraries of one of these families yielded a 14-amino acid
peptide with high affinity for the TPO receptor. When dimerized, the
resulting 28-amino acid peptide AF13948 was equipotent to TPO in
cell-based assays [21]. Subsequently, the sequence of AF13948 was
modified with amino acid substitution to yield AF15705, which was then
PEGylated to produce GW395058 (Fig. 1).
View larger version (14K):
[in this window]
[in a new window]
|
Figure 1. Amino acid sequences of AF15705 and GW395058.
AF15705 is a peptide dimer consisting of two identical amino acid
chains of 14 residues linked through their carboxyl termini to
the a and e amino groups of a lysine residue. The
sequence of AF13948 (see text) was modified by the replacement of an
alanine and a b-alanine residue with sarcosine (Sar) to
remove the chiral centers. Tryptophan residues were replaced with
napthylalanine (Npa) for ease of synthesis. The two resulting peptide
amino termini of AF15705 were conjugated to 20,000 MW polyethylene
glycol (PEG) to produce GW395058.
|
|
In vitro, GW395058 stimulates HuTPO receptor-transfected BaF3
cells and is equipotent to rHuTPO in stimulating megakaryocyte colony
formation [22]. In mice,
GW395058 has a long plasma half-life, elevates platelet counts, and
accelerates platelet recovery in a mouse myelosuppression model
[23]. In dogs, when
co-administered with granulocyte colony-stimulating factor, GW395058
has a long plasma half-life and accelerates platelet recovery in a dog
myelosuppression model (unpublished results; manuscript in
preparation). Preliminary studies of GW395058 in monkeys suggest that
repeated dosing with GW395058 does not cause an antibody response to
rHuTPO or AF15705 [24].
GW395058 shares no sequence homology with TPO but does compete
with TPO for binding to a common receptor site, raising the
possibility that a similarity in local structure could result in
shared epitopes. Thus, if GW395058 stimulated an antibody response
despite being PEGylated, anti-GW395058 antibodies could cross-react
with TPO. In this report, we evaluate: A) the immunogenicity of
GW395058 in mice after repeated dosing; B) the ability of rabbit
antibodies, elicited by immunizations with the non-PEGylated parent
peptide AF15705, to cross-react with rHuTPO, and C) the ability of
mouse anti-rHuTPO antibodies, elicited by repeated dosing with rHuTPO,
to cross-react with AF15705.
|
Experimental
Procedures
|
|---|
Materials
rHuTPO was obtained from R&D Systems, Inc., Minneapolis,
MN. AF15705 and GW395058 were obtained from the Biotechnology Product
Development Division, Glaxo Wellcome Inc., Research Triangle Park,
NC. All other chemicals were reagent grade or better.
GW395058 was prepared by derivatizing the two N-termini of the
AF15705 linear dimer with 20,000 molecular weight (MW) polyethylene
glycol (unpublished results; manuscript in
preparation). Derivatization of polyethylene glycol and its use for
preparing protein conjugates has been reviewed elsewhere [25, 26]. The MW of AF15705 and GW395058 are 3,295
and ~43,300, respectively. The amino acid sequences of AF15705 and
GW395058 are shown in Figure
1.
For the in vivo experiments described below, dosing solutions of
GW395058 or rHuTPO were prepared in phosphate buffered
saline. Solution concentrations of GW395058 were determined on the
basis of the extinction coefficient of the peptide
component. Concentrations or doses expressed in term of total mass of
PEGylated peptide would be ~13.5 ¥
greater.
Animal Handling, Dosing, and Sample
Collection
All animal procedures described in this report were approved by
the Institutional Animal Care and Use Committee and conducted in
accordance with federal guidelines.
Male BALB/c mice (five/group; three dose cycles/group) received
s.c. doses of rHuTPO (50 µg/kg; qd ¥ 5/cycle), GW395058 (25
µg/kg; single dose/cycle), or vehicle on Days 1, 43, and 85. Mouse
plasma samples for BIAcoreTM analysis were collected
following the third dosing cycle on day 6 and were stored frozen until
use. Blood (15 µl) samples for platelet count determinations were
collected from the tail vein after each cycle on day 6 and on days 20,
34, and 41 following the second cycle. Blood was diluted 1:50,000 in
isotonic saline prior to analysis. Mouse platelet counts were
determined with a Coulter Multisizer II (Coulter Electronics Limited;
Luton, Beds, UK).
Immunological Procedures
New Zealand white rabbits (3 to 5 kg) were initially immunized by
s.c. injections with 1.0 mg of an AF15705-bovine thyroglobulin
conjugate prepared by using standard glutaraldehyde coupling
procedures [27] and then
emulsified in Freund's complete adjuvant. Thereafter, rabbits
were administered s.c. injections of 0.1 mg antigen in Freund's
incomplete adjuvant at approximately two-week intervals for a one- to
two-month period. Blood was collected from anesthetized rabbits 7 to
10 days following immunization and the resulting antisera were stored
frozen until use.
Antigenicity Studies
To assess the immunogenicity of GW395058, samples of plasma from
BALB/c mice dosed with GW395058 or anti-AF15705 rabbit sera were
evaluated by using BIAcoreTM analysis. Plasma samples
were passed over immobilized rHuTPO or AF15705 to assess the presence
or absence of antibodies directed to rHuTPO or
AF15705. BIAcoreTM technology and its use in
characterizing inter-molecular interactions have been described
[28, 29]. The BIAcoreTM
2000 system, CM5 sensor chips, P-20 surfactant, and the coupling kit
which contained N-hydroxysuccinimide,
N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide, and ethanolamine
hydrochloride (pH 8.5) were purchased from BIAcoreTM
AB, Uppsala, Sweden. All other chemicals were reagent grade.
The rHuTPO was immobilized to the carboxyl groups of the
BIAcoreTM CM5 sensor chip hydrogel matrix by
activating the matrix with a mixture of 50 mM N-hydroxysuccinimide and
200 mM N-ethyl-N'-(3-diethylaminopropyl)-carbodiimide for 7
min. The rHuTPO (0.33 mg/ml) was diluted 1:10 in 10 mM sodium acetate
(pH 5), then injected onto the sensor chip for 3 min at a flow rate of
5 µl/min. Unreacted groups were then deactivated with a 7-min
injection of 1 M ethanolamine hydrochloride (pH 8.5). Similarly,
AF15705 was immobilized to BIAcoreTM sensor chips by
activating the chip for 12 min and then applying a peptide solution
(prepared by diluting a 1 mg/ml peptide stock 1:5) in 50 mM HEPES (pH
8) containing 1 M NaCl for 20 min. Immobilization was followed by a
deactivation step for 12 min. The surfaces of the chips were
regenerated after each binding cycle with 100 mM HCl. The
BIAcoreTM running buffer used for immobilization and
binding determinations contained 10 mM HEPES (pH 7.4), 150 mM NaCl,
3.4 mM EDTA, and 0.005% P-20
surfactant.
Histopathologic Evaluation of Mouse
Tissues
Six days after receiving the last doses of rHuTPO, GW395058, or
vehicle, mice were euthanized and necropsied. The following tissues
were collected, processed by routine histologic methods [30, 31], stained with HE, and examined
microscopically by an ACVP-certified veterinary pathologist: adrenal
gland (two sections), aorta, brain (three sections), cecum, epididymis
(two sections), esophagus, eye (two sections), femur, gallbladder,
harderian gland, heart, kidney (two sections), large intestine (two
sections), liver (two sections), lung (two sections), lymph nodes
(mesenteric and cervical), pancreas, parathyroid gland, pituitary
gland, prostate gland, salivary gland, sciatic nerve, seminal vesicle
(two sections), skeletal muscle, skin, small intestine (three
sections), spinal cord (three sections), spleen, sternum, stifle
joint, stomach (two sections), testis (two sections), thymus, thyroid
gland, tibia, tongue, trachea, and urinary
bladder.
|
Results
|
|---|
Immunoreactivity
of BALB/c Mouse Plasma Samples with rHuTPO or AF15705
Plasma samples (dose cycle three) from BALB/c mice that received
rHuTPO, GW395058, or vehicle over a three-month period were examined
by BIAcoreTM analysis for the presence of antibody
responses to rHuTPO or AF15705 (Fig. 2).
View larger version (15K):
[in this window]
[in a new window]
|
Figure
2. Antibody responses in mice to rHuTPO or GW395058.
Average mouse antibody responses (RU) to rHuTPO, GW395058, or
control are shown. Plasma samples were obtained from BALB/c mice
(five/group) on post-dose Day 6 (dosing cycle three) following
s.c. administration of rHuTPO at 50 µg/kg, qd ¥ 5/cycle (white
bar); GW395058 at 25 µg/kg, single dose/cycle (black bar); or
vehicle (gray bar).
|
|
No antibody binding was observed to either rHuTPO or AF15705 in
plasma samples from mice dosed for three cycles with GW395058 as
compared to the vehicle control. Mice dosed with rHuTPO showed marked
antibody responses to rHuTPO; however, no cross-reactivity with
GW395058 was observed in plasma samples from mice dosed with
rHuTPO. Mice in the rHuTPO group also showed diminished platelet
counts as compared to controls at cycle three with no thrombocytosis
in response to rHuTPO administration ( Fig. 3). In contrast, mice dosed with GW395058
showed a three- to fourfold elevation in circulating platelet counts
after each dose throughout the
study.
View larger version (14K):
[in this window]
[in a new window]
|
Figure
3. Platelet responses in BALB/c mice to rHuTPO or
GW395058. Average platelet counts in mouse blood samples
are shown. Blood samples were obtained from BALB/c mice (five/group;
three dose-cycles/group) receiving s.c. doses on Days 1, 43, and 85
(arrows) of rHuTPO at 50 µg/kg, qd ¥ 5/cycle (M); GW395058 at
25 µg/kg, single dose/cycle (D); or vehicle
(V)).
|
|
Histopathologic Findings in Mice
Light microscopic findings related to treatment with GW395058 or
rHuTPO were observed in multiple tissues. Tissue changes related to
treatment with GW395058 included: A) increased numbers and size of
megakaryocytes in the bone marrow and spleen ( Fig. 4); B) occurrence of a few megakaryocytes
in the liver and lung; C) reduced erythropoiesis in the bone marrow
( Fig. 5), and D)
increased erythropoiesis in spleen and liver.
View larger version (14K):
[in this window]
[in a new window]
|
Figure 4. Photomicrographs of HE-stained spleen sections from mice
given three dose cycles of vehicle, GW395058, or rHuTPO. Panel
1. Vehicle control. A few small megakaryocytes are
present (arrows). Bar = 200 µm. Panel 2.
GW395058 (25 µg/kg; single dose/cycle). Numerous large
megakaryocytes (arrows) and islands of erythropoietic cells (circle)
are present. Bar = 200 µm. Panel 3. rHuTPO
(50 µg/kg, qd ¥ 5/cycle). Megakaryocytes are absent. Bar = 200
µm.
|
|
View larger version (102K):
[in this window]
[in a new window]
|
Figure 5. Photomicrographs of HE-stained spleen sections from mice
given three dose cycles of vehicle, GW395058, or rHuTPO. Panel
1. Vehicle control. Megakaryocytes are scattered within
the marrow (arrows). Bar = 100 µm. Panel 2.
GW395058 (25 µg/kg; single dose/cycle). Megakaryocytes are
numerous and large, consuming much of the marrow space. Both nuclear
and cytoplasmic volumes are increased. Bar = 100 µm.
Panel 3. rHuTPO (50 µg/kg; qd ¥
5/cycle). Megakaryocytes are scarce. Bar = 100
µm.
|
|
Tissue changes related to treatment with rHuTPO included: A)
B-lymphocytic hyperplasia in lymphoid organs (spleen and cervical
lymph node), characterized by proliferation of immunoblastic
lymphocytes and increased numbers of plasma cells ( Fig. 4), and B) reduced numbers of
megakaryocytes in bone marrow (Fig. 5).
Immunoreactivity/Cross-Reactivity
of Artificially Generated Antibodies to AF15705 with rHuTPO
The immunoreactivity and cross-reactivity of rabbit anti-AF15705
sera to either AF15705 or rHuTPO were tested by
BIAcoreTM analysis. The averaged results from 15
individual rabbits are shown in Figure 6. All of the antisera showed strong reactivity
with AF15705. None of the antisera showed cross-reactivity with rHuTPO
above background control
levels.
View larger version (98K):
[in this window]
[in a new window]
|
Figure 6. Cross-reactivity of rabbit anti-AF15705 sera to rHuTPO or
GW395058. Average rabbit (n = 15) antibody
responses (RU) to rHuTPO (white bar), GW395058 (black bar), or control
(gray bar) are shown. Antisera were produced by immunization with an
AF15705-bovine thyroglobulin conjugate.
|
|
|
Discussion
|
|---|
Recently, patients participating in cancer and in platelet donor
clinical trials with PEG-rHuMGDF showed evidence of TPO-neutralizing
antibodies, and, as a result, the development of PEG-rHuMGDF was
discontinued [20]. The
production of TPO-neutralizing antibodies by repeated dosing with
PEG-rHuMGDF was unanticipated because prior work has shown that
PEGylated proteins often exhibit reduced immunogenicity in vivo
[26, 32-35].
GW395058, a PEGylated TPO peptide mimetic, shares no sequence
homology with TPO. However, GW395058 does compete with TPO for binding
to a common receptor [22, 23]. For this reason, studies were initiated to
rule out the possibility that conformational similarities in local
structure between GW395058 and TPO at the receptor might lead to the
production of TPO-neutralizing antibodies. In preliminary studies of
GW395058 immunogenicity, no antibody response was observed to AF15705
or rHuTPO in monkeys dosed repeatedly with GW395058 over a three-month
period [24].
Here we show that no antibody response to rHuTPO or AF15705 (the
non-PEGylated peptide parent of GW395058) occurred in plasma samples
from mice dosed for three cycles with GW395058. In contrast, mice
dosed with rHuTPO showed a marked antibody response to rHuTPO, but no
murine anti-rHuTPO antibody cross-reactivity with
AF15705. Hematological analysis of blood samples from mice dosed with
rHuTPO showed diminished platelet counts as compared to controls at
cycle three, with no thrombocytosis in response to rHuTPO
administration. This observation is consistent with an active
anti-rHuTPO antibody response. In contrast, mice dosed with GW395058
showed a three- to fourfold elevation in circulating platelet counts
after each dose of the compound throughout the three dosing cycles in
this study. This result is consistent with an absence of an antibody
response to either GW395058 or endogenous mouse TPO. No
histopathologic evidence of antigenic stimulation was observed by
microscopic examinations of tissue samples from mice dosed repeatedly
with GW395058, while megakaryocytopoiesis in marrow and extramedullary
sites was stimulated. In contrast, rHuTPO caused B-lymphocytic
hyperplasia in lymphoid tissues, consistent with a response to
antigenic stimulation, and suppressed megakaryocytopoiesis in
marrow. These pathologic findings suggest that repeated injections of
rHuTPO induced antibodies that not only neutralized the injected
rHuTPO and prevented it from stimulating megakaryocytopoiesis, but
also cross-reacted with endogenous murine TPO to suppress
megakaryocytopoiesis.
Rabbit antibodies elicited by immunizations with a thyroglobulin
conjugate of the non-PEGylated parent peptide AF15705 failed to
cross-react with rHuTPO. While anti-AF15705 sera from all 15 rabbits
reacted strongly with AF15705 and GW395058, none of the rabbit
antisera cross-reacted with rHuTPO.
Like rHuTPO and PEG-rHuMGDF, GW395058 is a TPO receptor agonist
that stimulates platelet production. Unlike rHuTPO and PEG-rHuMGDF,
GW395058 is a PEGylated small MW peptide that shares no sequence
homology with endogenous TPO. Thus, recognition of GW395058 by the
immune surveillance system is minimized. While it is not possible to
predict with certainty the immunogenicity of GW395058 in humans from
studies in animals, the data presented here suggest that the potential
for an immune response to GW395058 in humans will be low. Furthermore,
our data suggest that if an immune response to GW395058 were elicited
during clinical trials with this compound, such antibodies would be
unlikely to cross-react with endogenous human TPO.
|
ACKNOWLEDGMENT
|
|---|
We would like to thank Ms. Barbara Munch for necropsy and
histotechnical assistance, Mr. Chadwick T. Merrell for
Coulter analysis, and Mrs. Suzan de Serres and Dr. Joseph
L. Woolley for their editorial assistance.
|
References
|
|---|
-
Kudoh S, Yamada M. [Cytokine in cancer chemotherapy—clinical trials of IL-3, IL-11 and thrombopoietin against thrombocytopenia]. Gan To Kagaku Ryoho [Japanese Journal of Cancer & Chemotherapy]
1998; 25
:171
-176.
-
Isaacs C, Robert NJ, Bailey FA et al. Randomized placebo-controlled study of recombinant human interleukin-11 to prevent chemotherapy-induced thrombocytopenia in patients with breast cancer receiving dose-intensive cyclophosphamide and doxorubicin. J Clin Oncol
1997; 15
:3368
-3377.[Abstract/Free Full Text]
-
Tepler I, Elias L, Smith JW 2nd et al. A randomized placebo-controlled trial of recombinant human interleukin-11 in cancer patients with severe thrombocytopenia due to chemotherapy. Blood
1996; 87
:3607
-3614.[Abstract/Free Full Text]
-
Kaushansky K. Thrombopoietin: the primary regulator of platelet production (Comments). Blood
1995; 86
:419
-431.[Free Full Text]
-
Kato T, Ogami K, Shimada Y et al. Purification and characterization of thrombopoietin [published erratum appears in J Biochem (Tokyo) 1996;119:208]. J Biochem
1995; 118
:229
-236.[Abstract/Free Full Text]
-
Harker LA, Hunt P, Marzec UM et al. Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates. Blood
1996; 87
:1833
-1844a.[Abstract/Free Full Text]
-
Kabaya K, Akahori H, Shibuya K et al. In vivo effects of pegylated recombinant human megakaryocyte growth and development factor on hematopoiesis in normal mice. Stem Cells
1996; 14
:651
-660.[Abstract]
-
Akahori H, Shibuya K, Ozai M et al. Effects of pegylated recombinant human megakaryocyte growth and development factor on thrombocytopenia induced by a new myelosuppressive chemotherapy regimen in mice. Stem Cells
1996; 14
:678
-689.[Abstract]
-
Akahori H, Shibuya K, Obuchi M et al. Effect of recombinant human thrombopoietin in nonhuman primates with chemotherapy-induced thrombocytopenia. Br J Haematol
1996; 94
:722
-728.[Medline]
-
Shibuya K, Akahori H, Takahashi K et al. Multilineage hematopoietic recovery by a single injection of pegylated recombinant human megakaryocyte growth and development factor in myelosuppressed mice. Blood
1998; 91
:37
-45.[Abstract/Free Full Text]
-
Andrews RG, Winkler A, Myerson D et al. Recombinant human ligand for MPL, megakaryocyte growth and development factor (MGDF), stimulates thrombopoiesis in vivo in normal and myelosuppressed baboons. Stem Cells
1996; 14
:661
-677.[Abstract]
-
Molineux G, Hartley CA, McElroy P et al. Megakaryocyte growth and development factor stimulates enhanced platelet recovery in mice after bone marrow transplantation. Blood
1996; 88
:1509
-1514.[Abstract/Free Full Text]
-
Harker LA, Marzec UM, Hunt P et al. Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates. Blood
1996; 88
:511
-521.[Abstract/Free Full Text]
-
Harker LA, Marzec UM, Kelly AB et al. Prevention of thrombocytopenia and neutropenia in a nonhuman primate model of marrow suppressive chemotherapy by combining pegylated recombinant human megakaryocyte growth and development factor and recombinant human granulocyte colony-stimulating factor. Blood
1997; 89
:155
-165.[Abstract/Free Full Text]
-
Vadhan-Raj S, Murray LJ, Bueso-Ramos C et al. Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer (Comments). Ann Intern Med
1997; 126
:673
-681.[Abstract/Free Full Text]
-
Geissler K, Kabrna E, Stengg S et al. Recombinant human megakaryocyte growth and development factor increases levels of circulating haemopoietic progenitor cells post chemotherapy in patients with acute myeloid leukaemia. Br J Haematol
1998; 102
:535
-543.[Medline]
-
Basser RL, Rasko JE, Clarke K et al. Randomized, blinded, placebo-controlled phase I trial of pegylated recombinant human megakaryocyte growth and development factor with filgrastim after dose-intensive chemotherapy in patients with advanced cancer [published erratum appears in Blood 1997;90:2513]. Blood
1997; 89
:3118
-3128.[Abstract/Free Full Text]
-
Rasko JE, Basser RL, Boyd J et al. Multilineage mobilization of peripheral blood progenitor cells in humans following administration of PEG-rHuMGDF. Br J Haematol
1997; 97
:871
-880.[Medline]
-
Murray NA, Watts TL, Roberts IA. Endogenous thrombopoietin levels and effect of recombinant human thrombopoietin on megakaryocyte precursors in term and preterm babies. Pediatr Res
1998; 43
:148
-151.[Medline]
-
PJB Publications Ltd. Amgen Halts Growth Factor Trials. Scrip
1998; 2370
:20
.
-
Cwirla SE, Balasubramanian P, Duffin DJ et al. Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine. Science
1997; 276
:1696
-1699.[Abstract/Free Full Text]
-
Singer SC, Tansik RL, Dev IK et al. Pegylated thrombopoietin (TPO)-mimetic peptides bind human TPO receptor causing proliferation and maturation of megakaryocytes. Blood
1998; 92
:568a
.
-
Boytos CM, Rudolph SK, Merrell C et al. Pegylated thrombopoietin-mimetic peptides elevate blood platelets and ablate the chemotherapy-induced platelet nadir in mice. Blood
1998; 92
:377a
.
-
de Serres M, Hutchins J, Ellis B et al. Immunogenicity studies of the thrombopoietin mimetic peptide GW395058 in BALB/c mice, New Zealand white rabbits, and cynomolgus monkey. Blood
1998; 92
:173
.
-
Zalipsky S. Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates. Bioconjug Chem
1995; 6
:150
-165.[Medline]
-
Francis GE, Delgado C, Fisher D. PEG-modified proteins. In: Ahern TJ, Manning MC, eds. Stability of Protein Pharmaceuticals, Part B: In Vivo Pathways of Degradation and Strategies for Protein Stabilization, vol. 2. New York: Plenum Press, 1992:235-263.
-
Harlow E, Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1988.
-
Fagerstam LG, Frostell A, Karlsson R et al. Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping. J Mol Recognit
1990; 3
:208
-214.[Medline]
-
Fagerstam LG, Frostell-Karlsson A, Karlsson R et al. Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis. J Chromatogr
1992; 597
:397
-410.[Medline]
-
Sheehan DC, Hrapchak BB. Theory and Practice of Histotechnology. Columbus: Battelle Press, 1980.
-
Carson FL. Histotechnology, A Self-Instructional Text. Chicago: American Society of Clinical Pathologists, 1997.
-
Abuchowski A, McCoy JR, Palczuk NC et al. Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase. J Biol Chem
1977; 252
:3582
-3586a.[Abstract/Free Full Text]
-
Abuchowski A, van Es T, Palczuk NC et al. Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol. J Biol Chem
1977; 252
:3578
-3581.[Abstract/Free Full Text]
-
Saifer MG, Somack R, Williams LD. Plasma clearance and immunologic properties of long-acting superoxide dismutase prepared using 35,000 to 120,000 dalton poly-ethylene glycol. Adv Exp Med Biol
1994; 366
:377
-387.[Medline]
-
Clark R, Olson K, Fuh G et al. Long-acting growth hormones produced by conjugation with polyethylene glycol. J Biol Chem
1996; 271
:21969
-21977.[Abstract/Free Full Text]
accepted for publication June 14, 1999.
This article has been cited by other articles:
|
|
|
|
 
S. Frederickson, M. W. Renshaw, B. Lin, L. M. Smith, P. Calveley, J. P. Springhorn, K. Johnson, Y. Wang, X. Su, Y. Shen, et al.
A rationally designed agonist antibody fragment that functionally mimics thrombopoietin
PNAS,
September 26, 2006;
103(39):
14307 - 14312.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Drouet, F. Mourcin, N. Grenier, V. Leroux, J. Denis, J.-F. Mayol, P. Thullier, J.-J. Lataillade, and F. Herodin
Single administration of stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination soon after irradiation prevents nonhuman primates from myelosuppression: long-term follow-up of hematopoiesis
Blood,
February 1, 2004;
103(3):
878 - 885.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-H. Kie, W.-I. Yang, M.-K. Lee, T.-J. Kwon, Y.-H. Min, H.-O. Kim, H.-S. Ahn, S.-A. Im, H.-L. Kim, H.-Y. Park, et al.
Decrease in Apoptosis and Increase in Polyploidization of Megakaryocytes by Stem Cell Factor During Ex Vivo Expansion of Human Cord Blood CD34+ Cells Using Thrombopoietin
Stem Cells,
January 1, 2002;
20(1):
73 - 79.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. E. Greenwalt, J. Szabo, and I. Manchel
High Throughput Cell-Based Assay of Hematopoietic Progenitor Differentiation
J Biomol Screen,
December 1, 2001;
6(6):
383 - 392.
[Abstract]
[PDF]
|
|
|
|
|
|
|
G. D. Demetri
Targeted Approaches for the Treatment of Thrombocytopenia
Oncologist,
October 1, 2001;
6(2008):
15 - 23.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. C. Case, M. L. Hauck, R. L. Yeager, A. H. Simkins, M. de Serres, V. D. Schmith, J. E. Dillberger, and R. L. Page
The Pharmacokinetics and Pharmacodynamics of GW395058, a Peptide Agonist of the Thrombopoietin Receptor, in the Dog, a Large-Animal Model of Chemotherapy-Induced Thrombocytopenia
Stem Cells,
September 1, 2000;
18(5):
360 - 365.
[Abstract]
[Full Text]
|
|
|
Top
Abstract
Introduction
