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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2653-2659
Immunization With Recombinant Human Granulocyte-Macrophage
Colony-Stimulating Factor as a Vaccine Adjuvant Elicits Both a
Cellular and Humoral Response to Recombinant Human
Granulocyte-Macrophage Colony-Stimulating Factor
By
Douglas G. McNeel,
Kathy Schiffman, and
Mary L. Disis
From the Division of Oncology, University of Washington, Seattle, WA.
 |
ABSTRACT |
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an
important cytokine for the generation and propagation of
antigen-presenting cells and for priming a cellular immune response. We
report here that use of recombinant human GM-CSF (rhGM-CSF),
administered as an adjuvant in a peptide-based vaccine trial given
monthly by intradermal injection, led to the development of a T-cell
and antibody response to rhGM-CSF. An antibody response occurred in the
majority of patients (72%). This antibody response was not found to be
neutralizing. In addition, by 48-hour delayed type hypersensitivity
(DTH) skin testing, 17% of patients were shown to have a cellular
immune response to the adjuvant rhGM-CSF alone. Thymidine incorporation
assays also showed a peripheral blood T-cell response to rhGM-CSF in at
least 17% of the patients. The generation of rhGM-CSF-specific T-cell
immune responses, elicited in this fashion, is an important observation
because rhGM-CSF is being used as a vaccine adjuvant in various vaccine
strategies. rhGM-CSF-specific immune responses may be incorrectly
interpreted as antigen-specific immunity, particularly when local DTH
responses to vaccination are the primary means of immunologic
evaluation. We found no evidence of hematologic or infectious
complications as a result of the development of rhGM-CSF-specific
immune responses.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GRANULOCYTE-MACROPHAGE colony-stimulating
factor (GM-CSF) is a member of a large family of glycoprotein growth
factors that regulate the growth and differentiation of hematopoietic progenitor cells. Recombinant human GM-CSF (rhGM-CSF) has been used
extensively as a hematopoietic growth factor for patients with few side
effects. In several phase I studies, the adverse effects reported most
frequently were fever, chills, nausea, vomiting, asthenia, headache,
myalgias, arthralgias, and other nonspecific pain in the bones, chest
or abdomen.1,2 There has been one report of a localized
cutaneous reaction occurring at the site of rhGM-CSF injection in
breast cancer patients undergoing chemotherapy and receiving GM-CSF for
neutropenia.3 Other groups have reported the induction of a
transient antibody response to rhGM-CSF appearing after daily
administration, but without obvious adverse clinical sequelae.4-7 These systemic and immunologic effects were
observed after repetitive daily doses of rhGM-CSF over extended periods of time.
In addition to its activity as a hematopoietic growth factor, GM-CSF
acts at several levels in the generation and propagation of immune
responses. It is known to prime neutrophils for enhanced arachidonic
acid release and activate antibody-dependent cell-mediated cytotoxicity
of neutrophils, as well as act as a chemoattractant for eosinophils and
enhance the cytotoxicity of eosinophils.8,9 GM-CSF has also
been shown to induce the differentiation and promote the survival of
peripheral blood dendritic cells.10,11 These various
immunologic effects have led to several groups exploring the use of
GM-CSF as a vaccine adjuvant.
We have previously shown in a rodent model that GM-CSF can be used as a
vaccine adjuvant when mixed with a soluble antigen to induce both an
antibody and T-cell antigen-specific immune response.12
Other groups have transfected tumor cells with DNA encoding GM-CSF, or
directly injected GM-CSF into established tumors, and have shown
induction of antitumor immune responses in several murine
systems.13-16 Such results have led to phase I human
clinical trials using autologous GM-CSF gene-transduced, irradiated
tumors as vaccines in patients with metastatic renal cell
carcinoma17,18 and melanoma.19 Yet other groups
have used GM-CSF antigen fusion proteins as immunogens and showed
enhanced immunity to the antigen by such a method.20-22 The
administration of GM-CSF as a vaccine adjuvant is quite different from
the use of the cytokine as a hematopoietic growth factor.
We report here the generation of both a humoral and T-cell response to
rhGM-CSF in patients treated with soluble rhGM-CSF as a vaccine
adjuvant in a phase I peptide-based vaccine human clinical trial. In
previous reports of antibody responses to rhGM-CSF, the cytokine was
used on a daily basis. In our study, rhGM-CSF was administered monthly,
and both T-cell and antibody immune responses specific for rhGM-CSF
were generated after only a few vaccinations. This represents the first
report of a T-cell immune response to rhGM-CSF and the first report of
an immune response being generated to rhGM-CSF when used as a soluble
vaccine adjuvant.
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MATERIALS AND METHODS |
Detection of rhGM-CSF antibodies by enzyme-linked immunosorbent assay
(ELISA).
Human antibodies against rhGM-CSF were detected by indirect ELISA in
similar fashion to results previously published.5,23 In
brief, 1.0 µg/mL rhGM-CSF (Immunex Corp, Seattle, WA) in 50 mmol/L
sodium carbonate buffer (pH 9.6) was adsorbed to alternate wells of
Immulon-4 polystyrene plates (Dynex Technologies Inc, Chantilly, VA)
overnight at 4°C. Control wells contained sodium carbonate buffer
alone. Plates were then blocked with phosphate-buffered saline
(PBS)/1% bovine serum albumin (BSA) for 1 hour at room temperature,
and then washed with PBS/0.1% Tween-20. Patient sera was serially
diluted 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1,600, and 1:3,200 in
PBS/1% BSA and added to control (no rhGM-CSF) and experimental wells
(containing rhGM-CSF) for 1 hour at room temperature. After incubation,
plates were washed and an horseradish peroxidase (HRP)-conjugated sheep antihuman Ig antibody (Amersham,
Arlington Heights, IL), diluted 1:5,000 in PBS/1%BSA was added. After
an hour incubation at room temperature, the plates were washed and developed with tetramethylbenzidine (TMB) peroxidase
substrate (Kierkegard and Perry Laboratories, Gaithersburg, MD).
Reactions were stopped with addition of HCl to 0.5 N concentration, and the plates were then read at optical density (OD) 450 nm.
Data are reported as the  OD = OD450 (experimental well)-OD450
(control well).
Detection of rhGM-CSF antibodies by immunoblot.
A total of 0.1 µg rhGM-CSF produced in either yeast (Immunex Corp) or
Escherichia coli (E. coli) (Sigma, St Louis, MO), 0.1 µg ovalbumin, 0.1 µg lysozyme, and 10 lytic-forming units
(LFU) tetanus toxoid were resolved on 12% 1:19
bis:acrylamide polyacrylamide gels under nondenaturing conditions and
then transferred to Hybond-C nitrocellulose sheets (Amersham) by the
method of Towbin et al.24 The nitrocellulose sheets were
then blocked for 1 hour with PBS/5% BSA/1% IGEPAL CA-630
at room temperature. Patient sera was diluted 1:250 in PBS solution
(PBS/1% BSA/0.1% IGEPAL CA-630) and then used to probe the
nitrocellulose sheets overnight at 4°C. The sheets were then washed
with PBS solution and incubated for 1 hour at room temperature with an
HRP-conjugated sheep antihuman Ig detection antibody (Amersham) diluted
1:5,000 in PBS solution. After incubation with the secondary antibody,
the sheets were washed and developed with an ECL detection kit
(Amersham) according to the manufacturer's instructions and exposed to
radiographic film.
Detection of neutralizing antibodies to GM-CSF.
As a bioassay for the neutralizing effect of GM-CSF antibodies, the
GM-CSF-dependent human erythroleukemia cell line TF-1 (American Type
Culture Collection, Manassas, VA) was grown in RPMI 1640 (GIBCO, Grand
Island, NY) with 2 mmol/L L-glutamine, 1.5 g/L
NaHCO3, 4.5 g/L glucose, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 10% human A/B serum (ICN Flow, Costa Mesa, CA), and 5 ng/mL
rhGM-CSF (Immunex Corp).25 Cells were then washed
extensively and plated in sterile microtiter plates at 104
cells/well in 100-µL volumes in media without added rhGM-CSF, cell
growth being supportable with human serum. IgG was purified from
patients' sera using protein A chromatography (Avidchrome-Protein A
kit, Sigma), and resuspended at 200 µg/mL in media. Eight serial twofold dilutions of purified IgG were then added in 100-µL volumes to triplicate wells of TF-1 cells. Cells were cultured for 48 hours at
37°C in a humidified incubator and then pulsed with 1 µCi
3H-thymidine per well for 8 hours. Cells were then
harvested onto glass fiber filters, and the incorporated radioactivity
was measured with a Microbeta 1450 scintillation counter (Wallac,
Turku, Finland). Results are reported as the mean and standard
deviation of triplicate wells.
Detection of rhGM-CSF-specific T-cell immunity by
3H-thymidine incorporation assay.
Peripheral blood mononuclear cells (PBMC) were prepared by Ficoll-Paque
(Pharmacia AB, Uppsala, Sweden) centrifugation and resuspended in media
consisting of equal parts of EHAA 120 (Biofluids, Rockville, MD) and RPMI 1640 (GIBCO) with 10 mmol/L
L-glutamine, 2% penicillin/streptomycin, 50 µmol/L
 -mercaptoethanol, and 10% human AB serum (ICN Flow). Cells were
then analyzed for a T-cell proliferative response after exposure to
rhGM-CSF or ovalbumin, as a negative control protein, at varying
concentrations (0.25 µg to 1.0 µg/mL). A total of 2 × 105 PBMC/well were plated into 96-well round bottom
microtiter plates (Costar, Cambridge, MA) in triplicate cultures for
each concentration of protein tested and incubated with antigen at
37°C in an atmosphere of 5% CO2 for 5 days. Eight
hours before termination of culture, each well was pulsed with 1 µCi
3H-thymidine (New England Nuclear, Wilmington, DE). The
cultures were then harvested onto glass fiber filters, and the
incorporated radioactivity was measured as above. The results are
reported as the mean and standard deviation of triplicate assays.
Stimulation index (SI) is defined as the mean of experimental wells
divided by the mean of the control wells (no antigen).
Determination of delayed type hypersensitivity responses (DTH) to
rhGM-CSF.
At the end of the study, patients were skin tested against rhGM-CSF at
100 µg administered intradermally (i.d.) at a separate location from
the peptide vaccine site (on the back). A total of 100 µL sterile
water i.d. was used as a negative control. Induration was measured in
mm at 48 hours. Positive rhGM-CSF DTH sites, those  5 mm, were
biopsied with a 4-mm punch biopsy and submitted for immunohistochemical
staining (Phenopath Laboratories, Seattle, WA). Tissues were assayed
for expression of CD3, CD4, CD8, CD20, and CD1a.
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RESULTS |
Patients immunized with rhGM-CSF as an adjuvant may develop DTH
responses to rhGM-CSF.
A Phase I clinical trial of HER-2/neu peptide based vaccines is ongoing
at the University of Washington.26 Patients with HER-2/neu
overexpressing breast, ovarian, and nonsmall cell lung cancer are
vaccinated i.d. with peptides derived from the protein structure of the
HER-2/neu oncogenic protein and rhGM-CSF (purified from overexpression
in yeast, Immunex Corp) as a vaccine adjuvant (Disis et al,
submitted). Each vaccine tested is composed of three HER-2/neu peptides 15-18 amino acids in length.27 Patients
receive six monthly injections of peptide admixed with 250 µg
rhGM-CSF. At the time of this analysis, 18 patients have completed all
six vaccinations. During the course of vaccination, the majority of patients develop local DTH responses to the vaccine, and three patients
have developed immediate hypersensitivity responses at the site of
vaccination. As an example, one of these patients, CR4723, a woman with
stage IV breast cancer, received the first two monthly vaccinations
without any local erythema. After the third immunization, however, she
developed marked erythema and induration at the site of vaccination
within minutes of administration. No generalized erythema, urticaria,
or pulmonary symptoms were noted. A DTH response at the vaccine site
developed within 48 hours after injection. After the fourth and
subsequent vaccinations, similar local reactions were noted with
induration and erythema lasting up to 14 days after the vaccination.
After the final vaccination, formal DTH testing is performed on all
patients with the individual peptides derived from their vaccine,
rhGM-CSF alone, and sterile water as a control. The DTH testing is
administered i.d. at a site on the back distant from the original
vaccine site, and indurated reactions are read at 48 hours. At 48 hours, this particular patient had a DTH response of 5 × 6 mm to
rhGM-CSF. Likewise, she responded by DTH testing to one of the peptides
in her immunizing mix, with a 4 × 6 mm response to p369-384, but
no responses to either of the other two immunizing peptides, p688-703
or p971-984. Three of 18 patients (17%) immunized with rhGM-CSF in
this fashion have developed similar rhGM-CSF-specific DTH responses.
Patients immunized with rhGM-CSF as an adjuvant may develop T-cell
immunity to rhGM-CSF.
Four millimeter punch skin biopsies were obtained from the DTH sites of
the patient described above. Figure 1 shows
the immunohistochemical evaluation of the cellular infiltrate at the
rhGM-CSF DTH site. Figure 1A shows a marked dermal cellular infiltrate
that was not seen in a control skin biopsy from a noninvolved site (not
shown). Immunohistochemical staining (Fig 1B through F) shows a
predominant T-cell (CD3+) infiltrate with mixed
CD4+ and CD8+ lymphocytes. There is a marked
infiltration of CD1a+ cells, but relative absence of cells
bearing CD20.
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| Fig 1.
The rhGM-CSF-specific DTH response is a predominant
T-cell (CD3+) infiltrate with mixed CD4+
and CD8+ lymphocytes. (A) Hematoxylin and eosin staining
of the rhGM-CSF-induced dermal cellular infiltrate (30x). (B through
F) Represents the immunohistochemical staining of the rhGM-CSF DTH site
for patient CR4723. (B) CD3 (30x); (C) CD4 (30x); (D) CD8 (30x); (E)
CD20 (40x); (F) CD1a (30x).
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In addition, three patients who have completed all six immunizations
have evidence of a peripheral blood T-cell response to rhGM-CSF
developing during the course of vaccinations. As an example, Fig 2 shows the results of a 5-day
3H-thymidine incorporation assay for a patient, CZ8474,
using PBMC collected at baseline before and then after the six
vaccinations. An SI 2.0 is consistent with an immunized response.
There was no evidence of a T-cell response to rhGM-CSF before
immunization (SI, 1.2). After six vaccinations, however, the T-cell
response to rhGM-CSF was significant and specific (SI, 6.3). No
response was detected to an irrelevant control antigen, ovalbumin (SI, 1.2). Similar results were found with two other patients, in which SIs
in response to rhGM-CSF of 2.6 and 6.8 were also obtained after
vaccination, but not before vaccination (data not shown).
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| Fig 2.
Patients immunized with rhGM-CSF as an adjuvant may
develop peripheral blood T-cell responses to rhGM-CSF. A total of 2 × 105 PBMC were incubated with increasing concentrations of
rhGM-CSF or ovalbumin as a negative control irrelevant antigen. At the
end of a 5-day incubation, cultures were pulsed for 8 hours with 1 µCi 3H-thymidine and the incorporated radioactivity
counted. The results are reported as the mean cpm and standard
deviation of triplicate wells.
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Patients immunized with rhGM-CSF as an adjuvant may develop an
antibody response to rhGM-CSF.
Sera were obtained from patients before vaccination and after six
vaccinations. Sera from the 18 patients who have completed all six
immunizations were analyzed by ELISA for development of antibodies to
rhGM-CSF. As an example, Fig 3A shows the
rhGM-CSF antibody responses for patients CR4723 and MT8463. Similar
analysis of the other 16 patients who have completed all six
vaccinations showed that 13 of the 18 patients (72%) who completed all
six immunizations developed novel or enhanced antibodies to GM-CSF during the course of the six vaccinations (data not shown). The majority of these responses was detectable at dilution titers ranging
from 1:25 to 1:1,600 and was of the IgG1 class (data not shown). The
development of rhGM-CSF-specific antibodies was confirmed by Western
blot analysis (Fig 3B). Moreover, the antibodies recognized not only
rhGM-CSF expressed in yeast (lanes 4), but also rhGM-CSF expressed in
E. coli (lanes 5).
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| Fig 3.
Patients immunized with rhGM-CSF as an adjuvant may
develop an antibody response to rhGM-CSF. (A) Serum antibody response
by ELISA for rhGM-CSF for two patients, CR4723 and MT8463, before
starting the vaccine study ( ) and after completing all six
immunizations ( ). (B) Western blot analysis of sera analyzed in (A).
Polyacrylamide gel lanes include 0.1 µg lysozyme (negative control),
0.1 µg ovalbumin (negative control), 10 LFU tetanus toxoid (positive
control), 0.1 µg yeast-expressed rhGM-CSF, 0.1 µg E. coli-expressed rhGM-CSF. Panels show the antibody responses
from the prevaccination sera of patients CR4723 and MT8463 and with the
sera obtained after six vaccinations.
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A growth inhibition assay of the GM-CSF-dependent erythroleukemia cell
line, TF-1, was performed with immunoglobulin purified from patients
pre- and postimmunization.25 That is, purified IgG was
cultured with the TF-1 cell line, and the growth inhibition measured as
a decrease in the amount of 3H-thymidine uptake after 48 hours in culture. No neutralizing activity was found in any patient
analyzed. As examples, Fig 4 shows the
results of neutralization assays using IgG purified from patients
CR4723 and MT8463 whose antibody responses were shown in Fig 3.
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| Fig 4.
Antibodies to rhGM-CSF generated in immunized patients
are not neutralizing in activity. IgG purified from patients CR4723 and
MT8463 preimmunization and after all six immunizations was diluted in
media and cultured with 104 TF-1 cells for 48 hours. At the
end of 48 hours, cultures were pulsed for 8 hours with 1 µCi
3H-thymidine and the incorporated radioactivity counted.
The results are reported as the mean cpm of triplicate wells.
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DISCUSSION |
GM-CSF and other cytokines that can influence the generation of immune
responses are increasingly being explored as vaccine adjuvants.28-30 Immunologic cytokines, themselves, however,
may be immunogenic. It has been shown that immunocompetent and
immunocompromised patients treated with rhGM-CSF can develop an
antibody response to rhGM-CSF.4-6 Indeed, high-titer
antibodies to rhGM-CSF have been identified in human IgG preparations
from donors who have presumably never received treatment with
rhGM-CSF.31 Data presented here is the first report of
antibody and T-cell immunity directed against rhGM-CSF developing in
patients who have received soluble rhGM-CSF as an adjuvant in the
course of a vaccine trial. The generation of rhGM-CSF-specific T-cell
immune responses elicited in this fashion is important for several
reasons: (1) rhGM-CSF-specific immune responses may incorrectly be
interpreted as antigen-specific immunity; (2) the development of
rhGM-CSF-specific immunity could potentially limit the use of the
cytokine as an adjuvant when multiple immunizations are used; (3)
theoretically, the development of significant rhGM-CSF-specific
immunity may limit the generation of an immune response to a weak tumor
antigen, such as "self" antigens targeted in cancer vaccines, and
finally; (4) the long-term effects of the generation of T-cell immunity
to rhGM-CSF are currently unknown.
GM-CSF is known to function at several levels in the generation of an
immune response, primarily because of its role in the growth and
differentiation of antigen-presenting cells. GM-CSF is known to be able
to promote the differentiation of progenitor cells to functional
dendritic cells32 with reversible increases in major
histocompatability complex (MHC) class II expression, CD1a expression,
and membrane-bound IL-1 on GM-CSF stimulation.33 Likewise,
GM-CSF has been shown to augment the expression of costimulatory molecules, CD80 and CD86, on dendritic cells and to presumably thereby
augment the antigen presentation function of these
cells.11,34 Furthermore, GM-CSF has been shown to be a
chemoattractant for antigen-presenting cells.35 All of
these functions have made GM-CSF a potentially interesting cytokine for
use as a vaccine adjuvant.28 We have previously shown that
GM-CSF, when mixed with a soluble antigen, can act as adjuvant in the
induction of both antigen-specific antibodies and an antigen-specific
T-cell response in a rodent model.12 Others have shown
GM-CSF used as an adjuvant can markedly enhance the immunogenicity of a
human hepatitis protein vaccine.36,37 Currently, we have a
phase I clinical trial underway using rhGM-CSF as an adjuvant for a HER-2/neu peptide vaccine for patients with advanced stage breast, ovarian, or nonsmall cell lung cancer whose tumors express HER-2/neu. In this trial, patients receive 250 µg rhGM-CSF admixed with each peptide vaccine administered monthly for 6 months.
We report here that in the 18 patients who have completed all six
vaccinations in our phase I trial, 17% (3 of 18) developed a DTH
response specific for rhGM-CSF; 72% (13 of 18) developed detectable
antibody responses to rhGM-CSF. These antibody responses were not found
to be neutralizing in TF-1 growth inhibition assays. Immunohistochemical analysis of these DTH sites showed infiltration of
a mixed CD4+/CD8+ T-cell population, as well as
CD1a+ antigen-presenting cells. Likewise, thymidine
incorporation assays for at least three of the patients showed a
significant CD4+ T-cell proliferation in the presence of
rhGM-CSF.
DTH responses are often used as a measure of the immunity generated to
an antigen during the course of immunization.38 Our observations suggest that one should be cautious in interpreting DTH
responses at the vaccination site where rhGM-CSF is used as an adjuvant
because an immune response to rhGM-CSF can itself result in a positive
DTH response. This is particularly important for vaccine trials in
which GM-CSF-transduced tumors or GM-CSF fusion proteins are used, as
it is difficult to separate the "adjuvant" from the
"antigen", and rhGM-CSF-specific immune responses may incorrectly attributed to antigen-specific immunity.
The observation that rhGM-CSF can induce an immune response suggests
that it may limit the use of the cytokine when multiple immunizations
are used. Others have shown that the development of an antibody
response to rhGM-CSF, in patients treated with rhGM-CSF as a
hematopoietic growth factor, correlated with decreased GM-CSF serum
levels and decreased effectiveness of rhGM-CSF in augmenting the
peripheral white blood cell count.7 It remains a
theoretical risk, therefore, that the development of immunity to
rhGM-CSF could limit its effectiveness as a vaccine adjuvant, simply by
rapid clearing of the adjuvant. This could be important in immunization
strategies such as this one in which multiple immunizations are
required. We have not, however, observed any correlation between
development of immunity to rhGM-CSF and lack of the development of
immunity to the antigen HER-2/neu. Likewise, patients on study have
been followed monthly with respect to hematologic parameters, and no
patients who developed either rhGM-CSF antibody or T-cell immune
responses have had any appreciable decrease in peripheral white blood
cell counts.
The development of a significant rhGM-CSF immune response could
theoretically limit the generation of immunity to "weak"
antigens, such as "self" antigens targeted in cancer vaccines.
Chen and Levy23 have reported that multiple immunizations
with one GM-CSF antigen fusion construct in a murine model did result
in an antibody response that abrogated the response to subsequent
vaccinations with another GM-CSF-antigen fusion construct. However,
this was not the case in our model. In our current study, the
development of immunity to rhGM-CSF is not associated with inability to
immunize to HER-2/neu-derived peptides.
The observation of an immune response being generated to a "self"
cytokine adjuvant is not necessarily surprising. The development of
antibodies to different preparations of insulin, which is administered subcutaneously, has long been a problem in the treatment of diabetes. Likewise, others have shown that the development of neutralizing antibodies to interferon- has limited treatment with this
cytokine.39,40 The rhGM-CSF used in these studies is a
recombinant protein produced in a yeast system.41 The
protein differs from the human protein at a single amino acid and is
differentially glycosylated. These differences may be enough to
generate a cross-reactive immune response to the native
protein.7 We have shown that the antibody response produced
also recognizes rhGM-CSF expressed in E. coli, suggesting that
the antibody response is directed at the amino acid backbone of GM-CSF,
as the E. coli protein is not glycosylated. Other groups have
shown that daily treatment with rhGM-CSF as a hematopoietic growth
factor can result in the transient production of neutralizing
antibodies to human GM-CSF with biologic effects.4,5,7 We
do not find neutralizing activity of these antibodies in a TF-1
bioassay and have seen no clinical adverse outcomes such as decreases
in peripheral white blood cell counts. Moreover, the presence of
high-titer antibodies to rhGM-CSF in normal blood donors31
suggests that such an immune response is compatible with normal
hematopoeisis. The data presented here, however, is the first report of
a T-cell response being generated to rhGM-CSF. The long-term
implications of such an immune response are currently unknown. While
antibody responses generated against rhGM-CSF have previously been
shown to resolve 30 weeks after treatment,4 it is not yet
known how durable the antibody or T-cell responses are after
administration of rhGM-CSF by a monthly i.d. vaccination route. The
durability and hematologic consequences of T-cell and antibody immunity
directed against GM-CSF will be evaluated in the long-term follow-up of
our patients who have received monthly i.d. rhGM-CSF as a soluble
vaccine adjuvant.
| |
ACKNOWLEDGMENT |
We thank the Immunex Corp for the gift of rhGM-CSF used as a vaccine
adjuvant in this study. We are grateful for the expert nursing care of
Fran O'Donnell, BSN. We acknowledge Marilyn Skelly and Dr Allen M. Gown of Phenopath Laboratories, Seattle, WA for the biopsy processing
and immunohistochemical staining of the skin biopsies. We also thank Dr
Michael Piepkorn for assistance in the pathologic assessment of the DTH response.
| |
FOOTNOTES |
Submitted July 22, 1998; accepted November 30, 1998.
Supported by Grant No. P32 CA09515-14 from the National Institutes of
Health (NIH) (to D.G.M.) and by Grants No. K08 CA61834 and R01 CA75163
from the NIH, National Cancer Institute (NCI) (to M.L.D.).
Patient care was conducted through the Clinical Research Center
Facility at the University of Washington, which is supported through
Grant No. MO1-RR-00037 from the NIH.
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 Douglas G. McNeel, MD, PhD,
Division of Medical Oncology, 1959 NE Pacific St, Box 356527, University of Washington, Seattle, WA 98195-6527.
| |
REFERENCES |
1.
Vial T, Descotes J:
Clinical toxicity of cytokines used as haemopoietic growth factors.
Drug Saf
13:371, 1995[Medline]
[Order article via Infotrieve]
2.
Steward WP, Scarffe JH, Austin R, Bonnem E, Thatcher N, Morgenstern G, Crowther D:
Recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) given as daily short infusions  a phase I dose-toxicity study.
Br J Cancer
59:142, 1989[Medline]
[Order article via Infotrieve]
3.
Locker GJ, Simonitsch I, Mader RM, Warlamides E, Gnant MFX, Jakesz R, Rainer H, Steger GG:
Cutaneous side effects in breast cancer patients treated with cytostatic polychemotherapy and rh GM-CSF: Immune phenomena or drug toxicity?
Breast Cancer Res Treat
34:213, 1995[Medline]
[Order article via Infotrieve]
4.
Ragnhammar P, Friesen H-J, Frödin J-E, Lefvert A-K, Hassan M, Österborg A, Mellstedt H:
Induction of anti-recombinant human granulocyte-macrophage colony-stimulating factor (Escherichia coli-derived) antibodies and clinical effects in nonimmunocompromised patients.
Blood
84:4078, 1994[Abstract/Free Full Text]
5.
Wadhwa M, Bird C, Fagerberg J, Gaines-Das R, Ragnhammar P, Mellstedt H, Thorpe R:
Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy.
Clin Exp Immunol
104:351, 1996[Medline]
[Order article via Infotrieve]
6.
Thompson JA, Lee DJ, Kidd P, Rubin E, Kaufmann J, Bonnem EM, Fefer A:
Subcutaneous granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndrome: toxicity, pharmacokinetics, and hematological effects.
J Clin Oncol
7:629, 1989[Abstract]
7.
Gribben JG, Devereux S, Thomas NSB, Keim M, Jones HM, Goldstone AH, Linch DC:
Development of antibodies to unprotected glycosylation sites on recombinant human GM-CSF.
Lancet
335:434, 1990[Medline]
[Order article via Infotrieve]
8.
Vadas MA, Nicola NA, Metcalf D:
Human granulocyte-macrophage colony stimulating factor is a neutrophil activator.
Nature
314:361, 1985[Medline]
[Order article via Infotrieve]
9.
Weller PF:
Cytokine regulation of eosinophil function.
Clin Immunol Immunopathol
62:S55, 1992[Medline]
[Order article via Infotrieve]
10.
Markowicz S, Engleman EG:
Granulocyte-macrophage colony-stimulating factor promotes differentiation and survival of human peripheral blood dendritic cells in vitro.
J Clin Invest
85:955, 1990
11.
Larsen CP, Ritchie SC, Hendrix R, Linsley PS, Hathcock KS, Hodes RJ, Lowry RP, Pearson TC:
Regulation of immunostimulatory function and costimulatory molecule (B7-1 and B7-2) expression on murine dendritic cells.
J Immunol
152:5208, 1994[Abstract]
12.
Disis ML, Bernhard H, Shiota FM, Hand SL, Gralow JR, Huseby ES, Gillis S, Cheever MA:
GM-CSF: An effective adjuvant for protein and peptide based vaccines.
Blood
88:202, 1996[Abstract/Free Full Text]
13.
Sanda MG, Ayyagari SR, Jaffee EM, Epstein JI, Clift SL, Cohen LK, Dranoff G, Pardoll DM, Mulligan RC, Simons JW:
Demonstration of a rational strategy for human prostate cancer gene therapy.
J Urol
151:622, 1994[Medline]
[Order article via Infotrieve]
14.
Thompson RC, Pardoll DM, Jaffee EM, Ewend MG, Thomas MC, Tyler BM, Brem H:
Systemic and local paracrine cytokine therapies using transduced tumor cells are synergistic in treating intracranial tumors.
J Immunother Emphasis Tumor Immunol
19:405, 1996[Medline]
[Order article via Infotrieve]
15.
Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, Jackson V, Hamada H, Pardoll D, Mulligan RC:
Vaccination with irradiated tumor cells engineered to secrete muring granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity.
Proc Natl Acad Sci USA
90:3539, 1993[Abstract/Free Full Text]
16.
Kurane S, Arca MT, Aruga A, Krinock RA, Krauss JC, Chang AE:
Cytokines as an adjuvant to tumor vaccines: Efficacy of local methods of delivery.
Ann Surg Oncol
4:579, 1997[Medline]
[Order article via Infotrieve]
17.
Simons JW, Jaffee EM, Weber CE, Levitsky HI, Nelson WG, Carducci MA, Lazenby AJ, Cohen LK, Finn CC, Clift SM, Hauda KM, Beck LA, Leiferman KM, Owens AH Jr, Piantadosi S, Dranoff G, Mulligan RC, Pardoll DM, Marshall FF:
Bioactivitiy of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer.
Cancer Res
57:1537, 1997[Abstract/Free Full Text]
18.
Berns AJ, Clift S, Cohen LK, Donehower RC, Dranoff G, Hauda KM, Jaffee EM, Lazenby AJ, Levitsky HI, Marshall FF, Mulligan RC, Nelson WG, Owens AH, Pardoll DM, Parry G, Partin AH, Piantadosi S, Simons JW, Zabora JR:
Phase I study of non-replicating autologous tumor cell injections using cells prepared with or without GM-CSF gene transduction in patients with metastatic renal cell carcinoma.
Hum Gene Ther
6:347, 1995[Medline]
[Order article via Infotrieve]
19.
Ellem KA, O'Rourke MG, Johnson GR, Parry G, Misko IS, Schmidt CW, Parsons PG, Burrows SR, Cross S, Fell A, Li CL, Bell JR, Dubois G, Moss DJ, Good MF, Kelso A, Cohen LK, Dranoff G, Mulligan RC:
A case report: Immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy.
Cancer Immunol Immunother
44:10, 1997[Medline]
[Order article via Infotrieve]
20.
Tao MH, Levy R:
Idiotype/granulocyte-macrophage colony-stimulating factor fusion protein as a vaccine for B-cell lymphoma.
Nature
362:755, 1993[Medline]
[Order article via Infotrieve]
21.
Hakim I, Levy S, Levy R:
A nine-amino acid peptide from IL-1 augments antitumor immune responses induced by protein and DNA vaccines.
J Immunol
157:5503, 1996[Abstract]
22.
Wortham C, Grinberg L, Kaslow DC, Briles DE, McDaniel LS, Lees A, Flora M, Snapper CM, Mond JJ:
Enhanced protective antibody responses to PspA after intranasal or subcutaneous injections of PspA genetically fused to granulocyte-macrophage colony-stimulating factor or interleukin-2.
Infect Immun
66:1513, 1998[Abstract/Free Full Text]
23.
Chen TT, Levy R:
Induction of autoantibody responses to GM-CSF by hyperimmunization with an Id-GM-CSF fusion protein.
J Immunol
154:3105, 1995[Abstract]
24.
Towbin H, Staehelin T, Gordon J:
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications.
Proc Natl Acad Sci USA
76:4350, 1979[Abstract/Free Full Text]
25.
Wadhwa M, Bird C, Fagerberg J, Gaines-Das R, Ragnhammar P, Mellstedt H, Thorpe R:
Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy.
Clin Exp Immunol
104:351, 1996
26.
Disis ML, Grabstein KH, Sleath PR, Cheever MA:
HER-2/neu peptide vaccines elicit T cell immunity to the HER-2/neu protein in patients with breast and ovarian cancer.
Proc Am Soc Clin Oncol
17:97a, 1998
27.
Disis ML, Cheever MA:
HER-2/neu oncogenic protein: Issues in vaccine development.
Crit Rev Immunol
18:37, 1998[Medline]
[Order article via Infotrieve]
28.
Fagerberg J:
Granulocyte-macrophage colony-stimulating factor as an adjuvant in tumor immunotherapy.
Med Oncol
13:155, 1996[Medline]
[Order article via Infotrieve]
29.
Pardoll DM:
Paracrine cytokine adjuvants in cancer immunotherapy.
Annu Rev Immunol
13:399, 1995[Medline]
[Order article via Infotrieve]
30.
Nohria A, Rubin RH:
Cytokines as potential vaccine adjuvants.
Biotherapy
7:261, 1994[Medline]
[Order article via Infotrieve]
31.
Svenson M, Hansen MB, Ross C, Diamant M, Rieneck K, Nielsen H, Bendtzen K:
A ntibody to granulocyte-macrophage colony-stimulating factor is a dominant anti-cytokine activity in human IgG preparations.
Blood
91:2054, 1998[Abstract/Free Full Text]
32.
Sallusto F, Lanzavecchia A:
Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor .
J Exp Med
179:1109, 1994[Abstract/Free Full Text]
33.
Fischer H-G, Frosch S, Reske K, Reske-Kunz AB:
Granulocyte-macrophage colony-stimulating factor activates macrophages derived from bone marrow cultures to synthesis of MHC class II molecules and to augmented antigen presentation function.
J Immunol
141:3882, 1988[Abstract]
34.
Ahlers JD, Dunlop N, Alling DW, Nara PL, Berzofsky JA:
Cytokine-in-adjuvant steering of the immune response phenotype to HIV-1 vaccine constructs.
J Immunol
158:3947, 1997[Abstract]
35.
Kaplan G, Walsh G, Guido LS, Meyn P, Burkhardt RA, Abalos RM, Barker J, Frindt PA, Fajardo TT, Celona R:
Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing.
J Exp Med
175:1717, 1992[Abstract/Free Full Text]
36.
Tarr PE, Lin R, Muellern EA, Kovarik JM, Guillaume M, Jones TC:
Evaluation of tolerability and antibody response after recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and a single dose of recombinant hepatitis B vaccine.
Vaccine
14:1199, 1996[Medline]
[Order article via Infotrieve]
37.
Hess G, Kreiter F, Kosters W, Deusch K:
The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on hepatitis B vaccination in haemodialysis patients.
J Viral Hepat
3:149, 1996[Medline]
[Order article via Infotrieve]
38.
Hadden JW:
T cell adjuvants.
Int J Immunopharmacol
16:703, 1994[Medline]
[Order article via Infotrieve]
39.
Steis RG, Smith JW, Urba WJ, Clark JW, Itri LM, Evans LM, Schoenberger C, Longo DL:
Resistance to recombinant interferon alfa-2a in hairy-cell leukaemia associated with neutralizing anti-interferon antibodies.
N Engl J Med
318:1409, 1988[Abstract]
40.
Antonelli G, Currenti M, Turriziani O, Dianzani F:
Neutralizing antibodies to interferon-: Relative frequency in patients treated with different interferon preparations.
J Infect Dis
163:882, 1991[Medline]
[Order article via Infotrieve]
41.
Cantrell MA, Anderson D, Cerretti DP, Price V, McKereghan K, Tushinski RJ, Mochizuki DY, Larsen A, Grabstein K, Gillis S, Cosman D:
Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor.
Proc Natl Acad Sci USA
82:6250, 1985[Abstract/Free Full Text]
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