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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3250-3258
Abrogation of the Hematological and Biological Activities of the
Interleukin-3/Granulocyte-Macrophage Colony-Stimulating Factor
Fusion Protein PIXY321 by Neutralizing Anti-PIXY321 Antibodies in
Cancer Patients Receiving High-Dose Carboplatin
By
Langdon L. Miller,
Edward L. Korn,
Diane S. Stevens,
John E. Janik,
Barry L. Gause,
William C. Kopp,
Jon T. Holmlund,
Brendan D. Curti,
Mario Sznol,
John W. Smith II,
Walter J. Urba,
Sarah E. Donegan,
Thelma M. Watson, and
Dan L. Longo
From the Frederick Cancer Research and Development Center, Biological
Response Modifiers Program, National Cancer Institute, Frederick, MD;
the Biometric Research Branch, Cancer Therapy Evaluation Program,
National Cancer Institute, Bethesda, MD; the Clinical Services Program,
SAIC Frederick, Frederick, MD; Immunex Research and Development Corp,
Seattle, WA.
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ABSTRACT |
This dose-escalation study was performed to evaluate the hematologic
activity, biological effects, immunogenicity, and toxicity of PIXY321
(an interleukin-3/granulocyte-macrophage colony-stimulating factor
fusion protein) administered after high-dose carboplatin (CBDCA)
treatment. Patients with advanced cancers received CBDCA at 800 mg/m2 intravenously on day 0 of repeated 28-day cycles. In
part A of the study, patients were treated with CBDCA alone during
cycle 1 and then received PIXY321 on days 1 through 18 of cycle 2 and later cycles. In part B, patients received 18 days of PIXY321 beginning
on day 1 of all CBDCA cycles, including cycle 1. PIXY321 was
administered subcutaneously in 2 divided doses. Total doses of 135, 250, 500, 750, and 1,000 µg/m2/d were administered to
successive cohorts of 3 to 6 patients in part A. In part B, patient
groups received PIXY321 doses of 750, 1,000, and 1,250 µg/m2/d. The hematologic effects of PIXY321 were assessed
in the first 2 cycles of therapy. Anti-PIXY321 antibody formation was
assessed by enzyme-linked immunosorbent assay (ELISA) and
neutralization assay. Of the 49 patients enrolled, 31 were fully
evaluable for hematologic efficacy. When comparing the first B cycle
(cycle B-1; with PIXY321) with the first A cycle (cycle A-1; without PIXY321), the fusion protein had no significant effect on platelet nadirs or duration of platelets less than 20,000/µL but was able to
speed the time of recovery of platelet counts to 100,000/µL (15 v 20 days; P = .01). Significant improvements in
neutrophil nadir and duration of ANC less than 500 were observed in
cycles A-2 and B-1 (with PIXY321) as compared with cycle A-1 (without PIXY321). Initial PIXY321 prophylaxis (cycle A-2 and cycle B-1), enhanced the recovery of ANC to greater than 1,500/µL by an average of at least 8 days as compared with cycle A-1 (without PIXY321; P  .004). However, positive PIXY321 hematologic effects were lost in the second course of PIXY321 among patients treated in part B. ELISA analysis showed that 92% of patients had developed neutralizing
anti-PIXY321 antibodies by the completion of 2 PIXY321-containing cycles. The incidental action of PIXY321 to depress serum cholesterol levels was also abrogated during cycle B-2. We conclude that PIXY321 was active in speeding hematologic recovery but that neutralizing anti-PIXY321 antibody formation suppressed the hematologic and biochemical effects by the second cycle of PIXY321 administration. The
immunogenicity of this fusion protein provides a cautionary warning
that clinical development of bioengineered human molecules requires
thorough testing for immune neutralization.
This is a US government work. There are no restrictions on its use.
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INTRODUCTION |
THE MYELOTOXIC limitations on cytotoxic
drug delivery have spurred considerable interest in methods for
selectively ameliorating hematologic side effects. The frequency of
neutropenia as an oncologic problem has been the basis for granulocyte
colony-stimulating factor (G-CSF) and granulocyte-macrophage
colony-stimulating factor (GM-CSF) development. However, although these
agents have consistently enhanced recovery of neutrophils after
intensive chemotherapy, testing in randomized clinical trials has shown
that they do not beneficially alter the incidence or duration of
thrombocytopenia.1,2 Among a number of molecules with the
ability to enhance platelet production, the multipotential cytokine,
interleukin-3 (IL-3), has shown the capacity to promote in vitro growth
of megakaryocyte progenitors,3 and its clinical
administration has been associated with thrombocytosis.4,5
Bilineage stimulation of myelopoiesis and thrombopoiesis has been
increased by concurrent or sequential use of GM-CSF and IL-3
together.6-8
Based on such considerations, investigators at Immunex Corp (Seattle,
WA) constructed a fusion protein, designated PIXY321, that combines
GM-CSF and IL-3 into a single molecule.9 The component
cytokines are coupled via a flexible linker sequence so that the
binding domains of both moieties can interact with their independent
receptor binding sites.9 PIXY321 binds to cells via either
its GM-CSF or IL-3 domains, has comparable or greater affinity for
GM-CSF and IL-3 receptors in competitive binding studies, stimulates
the proliferation of normal human megakaryocyte and myeloid
progenitors, and induces bilineage platelet and neutrophil production
in monkeys.9-12
We hypothesized that PIXY321 might provide a conveniently administered
method of diminishing both the platelet and neutrophil toxicities
associated with chemotherapy. To test this hypothesis, we conducted
this dose-escalation trial of PIXY321 administered after intensive,
single-agent CBDCA. The objectives of the trial were to evaluate
patterns of myelosuppression and hematologic recovery associated with
the combined use of CBDCA and PIXY321, examine other biological
activities of PIXY321 administration, assess patients for the
production of anti-PIXY321 antibodies, and determine the toxicities of
the fusion protein.
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MATERIALS AND METHODS |
Patient eligibility and enrollment evaluation.
Patients greater than 18 years of age with metastatic solid tumors were
eligible for enrollment. They were required to have a Karnofsky
performance status  70%, relatively normal hemopoietic reserve (white
blood count [WBC] 4,000/µL, absolute neutrophil count [ANC]
1,500/µL, platelet count 100,000/µL, hemoglobin 8 g/dL),
estimated creatinine clearance greater than 70 mL/min, normal hepatic
function, and normal serum calcium. Patients with overt solid tumor
involvement of the bone marrow; previous pelvic radiotherapy or
irradiation to more than 20% of bone marrow; or earlier treatment with
CBDCA, nitrosoureas, or mitomycin-C were not eligible. A negative
 -HCG was required before study entry in all women of childbearing
potential. All patients were required to read and sign a consent form
approved by the local Institutional Review Board and by the Cancer
Therapy Evaluation Program (CTEP) of the National Cancer Institute.
Study drugs.
The study was sponsored by CTEP under BB-IND #4341. Glycosylated
PIXY321, produced in Saccharomyces cerevisiae, was provided via
CTEP by the Immunex Corp as a lyophilized powder. A lyophilized, investigational form of CBDCA (IND #19314) that is identical to commercially available CBDCA was also supplied by CTEP.
Treatment protocol.
At planned intervals of 28 days, all patients received a fixed dose of
CBDCA (800 mg/m2) by 30-minute intravenous infusion.
Patients enrolled to part A of the protocol received the first course
of CBDCA without PIXY321 support. During second and subsequent courses
of CBDCA, part A patients received CBDCA on day 0, followed by a course
of PIXY321 administered subcutaneously in divided doses approximately
every 12 hours beginning on day 1 and continuing for at least 18 days (Fig 1). Successive groups of 3 to 6 evaluable patients were enrolled to receive progressively higher doses
of PIXY321. Once the PIXY321 dose level was assigned for a particular
patient, that patient did not receive higher doses of PIXY321 in
subsequent cycles. Dose levels evaluated in part A of the protocol were
135, 250, 500, 750, and 1,000 µg/m2/d.
In part B of the trial, CBDCA was administered at a dose of 800 mg/m2 by 30-minute intravenous infusion every 28 days on
day 0 of each cycle. PIXY321 was initiated on day 1 and continued for
at least 18 days but was administered in every cycle, including the
first (Fig 1). PIXY321 doses of 750, 1,000, and 1,250 µg/m2/d were tested in part B.
Patients could continue on therapy until the occurrence of clinical or
radiographic evidence of tumor progression, unacceptable toxicity, or
withdrawal of patient consent. Dose reductions of CBDCA were not
allowed between cycles 1 and 2 unless a patient had experienced
nonhematologic CBDCA toxicity CTC grade 3 or a reduction in estimated
creatinine clearance to less than 60 mL/min. Subsequent therapy was
delayed until the ANC was greater than 1,500/µL and platelet count
increased to greater than 100,000/µL. While on protocol, patients
were not eligible to receive supportive therapy with other hematologic
growth factors, eg, G-CSF or GM-CSF, or nonphysiologic doses of
steroids. Bacterial infection was presumed in patients who developed a
fever of 38.5°C at a time when their ANC was less than 500/µL
and appropriate antibiotic intervention was provided. Depending on
availability, either 8 to 10 U of random-donor platelets or a
leukopheresis pack from a single donor was administered to subjects
each time that their platelet counts were less than 20,000/µL. Any
patient with a hemoglobin less than 8 g/dL received at least 2 U of
packed red blood cells.
Data monitoring.
Complete blood counts (CBCs) were obtained at least 3 times per week.
If the platelet count decreased to less than 50,000/µL, patients were
to have daily CBCs until the platelet count recovered to greater than
this level without transfusion. Serum was obtained on days 1, 2, 7, 14, and 28 for evaluation of serum chemistries. Part A patients had
antibody determinations obtained at baseline and at the end of cycle
A-2. Among part B patients, sera were tested at the end of cycles 1 and
2. Radiographic tumor evaluations were performed after every 2 cycles.
Serum antibody studies.
Enzyme-linked immunosorbent assays (ELISAs) for anti-PIXY321,
anti-IL-3, and anti-GM-CSF antibodies were performed at Immunex in
duplicate. Diluted patient sera were placed in plates coated with the
test cytokine. Goat antihuman IgG antibody conjugated to horseradish
peroxidase was used for signal detection. Positives were scored if the
sample results showed an optical density at least 4 times that of
matched baseline controls obtained before any CBDCA or PIXY321
administration. Sera that were positive for PIXY321 antibodies by ELISA
were tested for neutralizing antibody that prevented PIXY321-induced,
IL-3-induced, or GM-CSF-induced growth of human erythroleukemia
(TF-1) cells. Neutralization was considered present if the
posttreatment sample produced a 50% reduction in tritiated thymidine
incorporation as compared with that observed with the corresponding
baseline serum.
Statistical considerations.
Analysis of PIXY321 efficacy as a hematorestorative agent focused on
its effects on thrombocytopenia, neutropenia, and serum cholesterol
during the first 2 cycles of CBDCA administration. Comparisons were
made both within patients (cycle 1 v cycle 2) and between
patient groups (part A v part B). The focus of this analysis
was on patients who completed the first 2 courses of therapy
successfully; although this practice meant a reduction in the number of
patients analyzed, it was felt to be necessary to avoid bias resulting
from comparisons involving patients who were healthy enough to complete
2 courses of treatment and those who were not.
Parameters of interest in assessing PIXY321 hematologic effects were
the depth of the platelet and neutrophil nadirs, the duration of time
that the platelet counts were less than 20,000/µL and the ANC was
less than 500/µL, and the time to recovery of blood counts to values
that would allow CBDCA retreatment (platelets 100,000/µL, ANC
1,500/µL). On-time retreatment with CBDCA as well as incidence of
neutropenic fevers, bleeding episodes, and number of transfusions were
also assessed. Changes in serum cholesterol relative to baseline were
analyzed as an additional measure of PIXY321 biological effect.
Patients treated at all dose levels of PIXY321 were analyzed together
to increase the power of the tests, realizing that inclusion of
patients treated at lower PIXY321 dose levels might diminish the extent
of any differences in hematologic recovery. Paired comparisons were
made between the first and second courses of treatment in both parts A
and B of the study. Differences between groups A and B were assessed
with two-sample tests. Proportional differences between patient groups
and cycles were evaluated with two-sided, permutation exact tests.
Descriptive analyses of antibody formation were performed in all
patients who received PIXY321 and for whom sufficient serum was
available at any of the designated timepoints.
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RESULTS |
Patient characteristics.
Forty-nine patients (29 men and 20 women) were enrolled in the study.
The median age was 50 years (range, 24 to 71 years) and the median
Karnofsky performance status was 90% (range, 70% to 100%).
Thirty-four of the patients had undergone prior chemotherapy and 15 had
received previous radiotherapy. The predominant tumor types were
colorectal cancer (n = 15), melanoma (n = 12), renal cell (n = 7),
sarcoma (n = 4), lung (n = 3), and head and neck (n = 1). One
patient each with thymoma, carcinoid tumor, pheochromocytoma, and
cancers of the pancreas, breast, and adrenal glands were also enrolled.
Patient evaluability.
Of the 49 patients enrolled, 6 experienced progressive cancer and 2 requested removal from study after having received CBDCA alone during
cycle A-1. As a consequence, 41 patients actually received PIXY321 and
were evaluable for PIXY321 toxicity (24 patients in part A and 17 patients in part B). Ten other patients were not included in the
hematologic efficacy analysis. Reasons for exclusion were removal from
study for progressive disease (1 patient in part A and 3 patients in
part B) or excessive toxicity before completing 2 full CBDCA treatment
courses (1 patient in part A and 3 patients in part B). Protocol
violations were recognized in 2 part-A patients. One patient had a
combination of a rapidly declining performance status and excessive
prior cytotoxic therapy and the other received therapeutic G-CSF for
neutropenic sepsis. Thus, 20 patients who completed the entire course
of PIXY321 with full doses of CBDCA during both cycles A-1 and A-2 were
fully evaluable for hematologic effects (4 patients received 135, 5 patients received 250, 3 patients received 500, 4 patients received 750, and 4 patients received 1,000 µg/m2/d of PIXY321).
Eleven patients received full-dose CBDCA and a complete PIXY321 course
in both cycles B-1 and B-2 (4 patients received 750, 3 patients
received 1,000, and 4 patients received 1,250 µg/m2/d of
PIXY321). Twenty-four patients enrolled to part A and 15 patients
entered onto part B of the trial had sera available for antibody analysis.
Comparisons made both within evaluable patients (cycle 1 v
cycle 2) and between evaluable patient groups (part A v part B) indicated no significant differences in mean estimated creatinine clearance (P values for all comparisons .20) that might have been associated with confounding alterations in CBDCA clearance (data
not shown).
Hematologic efficacy.
Following the expected platelet nadir after administration of CBDCA
alone in cycle A-1, patients experienced a rebound thrombocytosis that
left the average platelet count significantly higher (P < .001) at the start of cycle A-2 than it had been before any therapy (Fig 2). Despite this boost in platelet
counts, a rapid decrease in their number during subsequent PIXY321
administration in cycle A-2 limited the ability of PIXY321 to improve
the nadir or shorten the period that platelet counts were less than
20,000/µL when cycle A-2 (with PIXY321) was compared with cycle A-1
(without PIXY321; Table 1). A greater
effect of PIXY321 on platelets seemed evident in the first-cycle
comparison of B-1 patients (with PIXY321) to those treated in A-1
(without PIXY321); platelet counts among B-1 patients decreased more
rapidly and then increased more quickly and to higher average levels
than among the A-1 controls, again with no significant effect on the
nadir. The use of higher PIXY321 dose levels and the lack of cumulative
marrow toxicity associated with prior CBDCA use may have accounted for
the more pronounced effect in cycle B-1 than in cycle A-2. Because the
predominant action of PIXY321 was to shift the platelet curves to the
left during cycle B-1, the time that counts were under 20,000/µL was not significantly altered. However, recovery of platelets was significantly enhanced; platelets reached 100,000/µL in 15 days with
PIXY321 during cycle B-1 as opposed to a 20-day recovery time in CBDCA
alone controls (P = .01).
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| Fig 2.
Platelet counts in patients completing 2 full cycles of
CBDCA. Curves depict results from 20 part A patients and 11 part B
patients.
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PIXY321 impact on neutrophil numbers was most conspicuous in the
initial, B-1 cycle of administration, in which marked elevations in ANC
were noted, peaking on day 4 (Fig 3). This
initial increase was followed by a blunted nadir and more rapid
neutrophil recovery. Also evident was a minor slump in average ANC
after discontinuation of PIXY321 on day 18; this was most obvious among
B-1 patients, but it is also apparent in those receiving PIXY321 for
the first time in cycle A-2. Significant improvements in nadir
neutrophil counts were noted with initial PIXY321 administration, both
in cycle A-2 or B-1 (with PIXY321) as contrasted with cycle A-1
(without PIXY321; Table 2). The initial
course of PIXY321 prophylaxis, whether given in cycle A-2 or cycle B-1
was also clearly able to hasten recovery of neutrophils, reducing the
duration of ANC less than 500/µL by 1.6 to 2.5 days and significantly
shortening the mean time required to achieve an ANC greater than
1,500/µL by at least 8 days (Table 2).
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| Fig 3.
ANC in patients completing 2 full cycles of CBDCA. Curves
depict results from 20 part A patients and 11 part B patients.
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When the fusion protein was administered for 2 successive cycles, it
proved unable to overcome the cumulative neutropenia associated with
repeated CBDCA administration; both the depth and duration of severe
neutropenia worsened in cycle B-2 as compared with cycle B-1 (Fig 3).
Although a modest rise in prenadir ANC was observed, the nadir was
significantly lower than seen in cycle B-1 (P = .008) and no
evidence of postnadir PIXY321 effect was observed. Particularly
striking was the protracted time in cycle B-2 between chemotherapy
administration and recovery of ANC to at least the 1,500 cells/µL
required for patients to initiate a third cycle of CBDCA. Cycle B-2
patients required a mean of 22 days to achieve this neutrophil level, a
period identical to the recovery time observed in cycle A-1 patients
receiving CBDCA alone (Table 2).
At least 1 platelet transfusion was administered to 65% (13/20) of
control subjects in cycle A-1 and the median platelet transfusion requirement for these patients was 1 transfusion per cycle. PIXY321 was
neither able to favorably decrease the proportion of patients needing
platelet transfusion nor able to reduce the median number of
transfusion events. No salutary effects on RBC transfusion requirements
were noted. One patient had a bleeding episode associated with a peptic
ulcer during cycle A-1. No bleeding occurred in cycles A-2, B-1, or
B-2. Neutropenic fever occurred in fewer than 18% of patients in any
cycle, and the incidence was not statistically altered by PIXY321 administration.
Platelet recovery did not delay administration of the next cycle of
CBDCA. When postponement of chemotherapy was required, failure to
attain an ANC of 1,500/µL was universally responsible. PIXY321,
administered for the first time in either cycle A-2 or B-1, reduced
treatment delays by enhancing neutrophil recovery. This could be
statistically significant; none (0/20) of the cycle A-2 patients failed
to recover on time while receiving PIXY321, whereas 30% (6/20) of
these same patients were unable to do so in cycle A-1 when not
receiving PIXY321 (P = .03). However, the loss of neutrophil
restorative activity observed with repeated administration of cytokine
prophylaxis caused 55% (6/11) of the patients in cycle B-2 to be
ineligible for chemotherapy on time on day 28; comparison with the 9%
(1/11) occurrence of delays in cycle B-1 (P = .06) or 0% in
A-2 (P = .001) indicated that this could be statistically worse.
Anti-PIXY321 antibodies.
The failure of PIXY321 to maintain its beneficial effects when
administered for more than a single treatment course led us to be
concerned that its activity was being suppressed immunologically. For
this reason, patient sera were analyzed by ELISA for the presence of
antibodies against PIXY321, IL-3, and GM-CSF. Samples that were
ELISA-positive were examined for the presence of neutralizing effects
on biologic function in vitro.
Greater than 80% of part B patients treated with PIXY321 were
ELISA-positive for anti-PIXY321 and anti-IL-3 antibodies by the end of
cycle 1 (Table 3). By the completion of
cycle B-2, 93% of patients had developed anti-PIXY321 antibodies, and,
in 92% of patients, this immune response proved to be neutralizing. Among patients treated in part B, 86% developed anti-IL-3 and 64%
had anti-GM-CSF antibodies by the end of cycle 2, although there was a
lesser likelihood that these would be neutralizing (29% and 45%,
respectively). There was the general sense that cycle A-2 patients,
some of whom received lower doses of PIXY321, were less likely to
develop neutralizing antibody; however, a formal regression analysis
failed to confirm a clear dose-response effect (data not shown). It is
also hypothetically possible that cumulative immunosuppression from the
chemotherapy may have blunted an antibody response in cycle A-2
patients, because they had received CBDCA twice before beginning their
initial PIXY321 course.
Other laboratory endpoints.
Having observed in vitro evidence of anti-PIXY321 antibody formation
and an in vivo reduction in PIXY321 effect on hematologic parameters,
we sought corroborating evidence of a decrease in other biological
activity. No alterations in lactate dehydrogenase, uric acid, or
alkaline phosphatase were noted as have been observed with
G-CSF13 or GM-CSF.14,15 However, significant
decrements in serum cholesterol (Fig
4) were noted, a finding consistent with known actions
of GM-CSF.14,15 The declines in serum cholesterol were most
pronounced when PIXY321 prophylaxis was administered for the first
time; in cycle A-2 or in cycle B-1, proportional decreases on days 2, 7, and 14 were significantly more profound during PIXY321
administration than those observed with CBDCA alone on the equivalent
days. However, consistent with the loss of fusion protein effect on
hematologic recovery during the second cycle of administration, the
PIXY321-induced depression of serum cholesterol was lost during cycle
B-2 at a time when most of these patients were known to have developed
in vitro neutralizing anti-PIXY321 antibodies; serum cholesterol levels
were statistically comparable to those observed during the CBDCA-alone
control cycle (A-1).
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| Fig 4.
Proportional serum cholesterol levels. Curves depict
results from 20 part A patients and 11 part B patients. Values marked
by an asterisk are significantly different from ( ) A-1 (-PIXY321) at
those timepoints (P .05, two-sided, permutation exact
tests).
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PIXY321 toxicity.
The most definite adverse PIXY321 event was low-grade skin irritation
at injection sites. Occasional patients also noted minor bruising at
injection sites during periods of thrombocytopenia. Generalized rashes
were not observed. The other common toxicities associated with PIXY321
administration were minor constitutional complaints of fever, chills,
myalgias, arthralgias, and headache that predominated in the first
several days of PIXY321 administration, primarily in the initial course
of treatment. Six serious adverse events were observed in patients
enrolled to the trial. Events felt not to be PIXY321-related included
an exacerbation of gout, acute renal failure, deep vein thrombosis, and
a fatal ischemic cerebral vascular accident. One patient had a
subendocardial infarction and another developed atrial fibrillation;
PIXY321 was listed as having a possible contributing role in these
conditions. Thirteen patients received chemotherapy beyond cycle 2 of
the protocol. None of these patients experienced any late or cumulative
PIXY321 toxicity; in particular, no allergic reactions or prolonged
neutropenia or thrombocytopenia occurred.
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DISCUSSION |
In this phase I trial of PIXY321, we attempted to characterize several
biological activities of PIXY321 over a range of doses that would
likely have positive hematologic activity and tolerable side effects
based on past experience with glycosylated forms of
IL-34,5,7 and GM-CSF.16-18
We found that, during the initial cycle of CBDCA, PIXY321 prophylaxis
was able to induce a more rapid recovery of platelet counts. However,
the depth of the nadir was less clearly affected by the cytokine, and
the time that platelet counts were less than 20,000/µL was not
altered because platelet counts decreased more quickly when the fusion
protein was administered. These relatively modest effects of PIXY321 on
platelet counts may have been presaged by the marginal evidence for
benefit that emerged from clinical trials using recombinant IL-3
prophylaxis after chemotherapy.19-21 Because it acts so
early in hematopoiesis and can stimulate such a broad variety of cell
lineages, the thrombocytosis induced by IL-3 occurs relatively
late5,22,23 and may be dampened by the counterregulatory
effects of other cell types and cytokines present in the hematopoietic
cascade. Evidence from canine studies indicates that GM-CSF stimulates
Kupffer-cell-mediated platelet destruction within the liver,
shortening platelet half-life24; such an effect may explain
the relatively rapid decrease in platelet counts that has been a
feature of PIXY321 prophylaxis25 and has also been seen
with administration of GM-CSF16,26 or
IL-3.27,28
Favorable actions on neutrophil numbers were much more evident with
PIXY321; significant improvements in both the depth and duration of
neutropenia were observed during the first course of fusion protein
administration, and these benefits enhanced the prospect that
subsequent chemotherapy could be administered on time. Unfortunately,
it became apparent during the B portion of the study, when patients
received PIXY321 prophylaxis after both first and second cycles of
CBDCA, that these advantages were not sustained. Neutrophil recovery
after the nadir was clearly worse late in cycle B-2 than during the B-1
cycle. Of particular note, the postnadir neutrophil recovery in cycle
B-2 was also less rapid than that observed in A-2 patients who received
PIXY321 for the first time in the second cycle. It appeared that the
second-cycle advantages of PIXY321 use were being abrogated only if the
fusion protein had been previously administered.
This loss of activity was most readily explained by immunologic
neutralization in patients who received 2 courses of the cytokine. The
timing of the loss of hematologic effects late in cycle B-2 was
consistent with in vivo induction of a primary immune response in cycle
B-1 that was boosted by later injection of antigen in the B-2 cycle. In
vitro, 53% of the patients had developed neutralizing antibodies by
the end of the B-1 cycle, and 92% of the participants had become
neutralizing-antibody positive at the completion of cycle B-2. The
correlation between the presence of in vitro neutralizing antibody and
loss of biological activity was strengthened by the finding that the
incidental actions of PIXY321 to lower serum cholesterol levels during
the first course of fusion-protein prophylaxis (cycles A-2 or B-1) were
also negated during cycle B-2. In vitro neutralization in PIXY321
assays appeared greater than in IL-3 and GM-CSF assays, suggesting that
the foreign linker portion of the molecule might have been involved in
inducing the immunogenic effect. However, specific studies to determine
the location of the immunogenic epitopes on the PIXY321 protein were
not performed, and it is not possible to exclude other reasons (eg, a
greater number of epitopes on the larger PIXY321 molecule) for the findings.
The formation of neutralizing antibodies clearly has precedent based on
past experience with other recombinant cytokines, eg,
interferon- 29 and IL-2.30,31 Ecogramostim
GM-CSF had also provoked antibody formation, both to itself and to
therapeutic monoclonal antibodies.32,33 Breaking of
tolerance to endogenous cytokines has been observed with G-CSF in an
experimental dog model34 and with pegylated recombinant
megakaryocyte growth and development factor (MGDF) in human
trials,35 resulting in neutropenia and thrombocytopenia, respectively.
Although we noted no overt clinical evidence that administration of
PIXY321 induced patients to break tolerance to endogenous GM-CSF or
IL-3, PIXY321 did appear to be potently immunogenic in this study. A
number of reasons may exist to explain this finding beyond the
artificial nature of PIXY321 and the fact that we very systematically
looked for antibody induction. It has become evident that GM-CSF has a
critical role in the proliferation, maturation, trafficking, and
function of antigen-presenting dendritic cells.36,37 It is
interesting to speculate that the GM-CSF moiety in PIXY321 might assist
dendritic cells in finding and processing the IL-3 or linker portions
of the fusion protein for presentation to lymphocytes. Such a strategy
has already been exploited preclinically to enhance the immunogenicity
of a tumor vaccine.38 Because relatively high concentration
of dendritic cells is found in the skin, the subcutaneous
administration of the drug twice per day in this trial may have
enhanced the likelihood of PIXY321 antigenic processing. It is also
possible that a high incidence of antibody induction was observed
because we treated patients with solid tumors who were better capable
of mounting an immune response; by contrast, individuals with lymphoid
malignancies might have relatively low rates of antibody induction to
recombinant cytokines.32
In other experience with PIXY321, patients received the cytokine after
very immunosuppressive treatment with high-dose alkylating agents,
steroids, or whole body irradiation,25,39-48 modalities that may be more immunosuppressive than CBDCA.49 The
contention that we did not induce substantial immune dysfunction with
administration of single-agent CBDCA may be supported by an Immunex
review of antibody formation in patients receiving PIXY321 in these
other studies; only 25% (76/302) developed anti-PIXY321 antibodies and in only 4.3% (13/302) was in vitro neutralization demonstrated (L. Garrison, personal communication, May 1996).
In conclusion, we have found that, although PIXY321 proved quite
tolerable over a wide range of doses when administered subcutaneously after high-dose CBDCA, it had only modest platelet restorative activity. In addition, the striking improvements in neutrophil accounts
observed with use of the PIXY321 were rapidly abrogated by the near
universal induction of neutralizing anti-PIXY321 antibodies. Ultimately, this experience with PIXY321 may represent an important warning that we must carefully assess the immunogenicity of new recombinant human hematopoietins and other proteins as they enter clinical trials. This may prove particularly important when the new
agent is a substantially modified version of a native molecule, immune
compromise is not likely to be present, the subcutaneous route is used,
and/or immune stimulatory molecules such as GM-CSF are administered concurrently.
| |
ACKNOWLEDGMENT |
The authors recognize the enormous contributions made to the completion
of this study by the nursing, laboratory, and administrative staffs of
the Biological Response Modifiers Program and the Clinical Services
Program of SAIC Frederick. The expert bibliographic assistance of
Carolyn Nagler is gratefully acknowledged. Our appreciation is also
extended to Nilsen L. Miller for diligence in collating study data.
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FOOTNOTES |
Submitted December 15, 1997; accepted December 14, 1998.
Preliminary results presented at the 12th (1993) and 15th (1996) Annual
Meetings of the American Society of Clinical Oncology, Orlando and the
8th Annual (1994) NCI-EORTC Symposium on New Drugs in Cancer Therapy,
Amsterdam, The Netherlands.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Langdon L. Miller, MD, Pharmacia & Upjohn,
Inc, Mail Code 7216-298-163, 7000 Portage Rd, Kalamazoo, MI 49001-0199;
e-mail: langdon.l.miller{at}am.pnu.com.
| |
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ABSTRACT
INTRODUCTION