1. No category

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1994 84: 4078-4087
Induction of anti-recombinant human granulocyte-macrophage
colony- stimulating factor (Escherichia coli-derived) antibodies and
clinical effects in nonimmunocompromised patients
P Ragnhammar, HJ Friesen, JE Frodin, AK Lefvert, M Hassan, A Osterborg and H Mellstedt
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Copyright 2011 by The American Society of Hematology; all rights reserved.
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Induction of Anti-Recombinant Human Granulocyte-Macrophage
Colony-Stimulating Factor (Escherichia coli-Derived) Antibodies
and Clinical Effects in Nonimmunocompromised Patients
By Peter Ragnhammar, Heinz-Jurgen Friesen, Jan-Erik Frodin, Ann-Kari Lefvert, Moustapha Hassan,
Anders Osterborg, and HAkan Mellstedt
The pharmacokinetics of recombinant human granulocytemacrophage colony-stimulating factor (rhGM-CSF), induction of anti-GM-CSF antibodies, and clinical effects related
t o the induction of the antibodies were analyzed in patients
with metastatic colorectal carcinoma (CRC)who were not on
chemotherapy (n = 20, nonimmunocompromisedpatients).
rhGM-CSF (250 pg/m2/d; Escherichia coli-derived) was administered subcutaneously for 10 days every month for 4
months. Eight patients with multiple myeloma (MM) on intensive chemotherapy followed by rhGM-CSF treatment
were also included (immunocompromisedpatients). After a
single injection of GM-CSF at the first cycle in CRC patients,
the maximum calculated concentration (Cmax)was 5.24 ?
0.56 ng/mL; the half life
was 2.91 f 0.8 hours; and the
area under the concentration curve (AUC) was 30.86 f 6.03
hours x ng/mL (mean f SE). No anti-GM-CSF antibodies
were detected. During the subsequent cycles, 95% of the
CRC patients developed anti-GM-CSF IgG antibodies,
which
significantly altered the pharmacokinetics of rhGM-CSF at
the third and fourth cycles with decreased, C
(2.87 f 0.57
ng/mL; P .05),(1.57
2 0.2 hours; P < .05), and AUC
(14.90 f 4.10 hours x ng/mL; P < .005). The presence of
anti-GM-CSF antibodies significantly reducedthe GM-CSFinduced enhancement of granulocytes, there
and was a clear
tendency for a decreased increment of monocytes. Antibodies diminished systemic sideeffects of rhGM-CSF. Only 1 of
8 MM patients showed a very low anti-GM-CSF antibody
titer after GM-CSF therapy, as shown by enzyme-linked immunosorbent assay and Westernblot. Therefore, in nonimmunocompromised patients,exogenous nonglycosylated
GM-CSF induced an anti-GM-CSF IgG antibody response
in practically all patients, which seemed t o be of clinical
significance. In immunocompromised patients, virtually no
significant antibody response was shown.
0 1994 by The American Society of Hematology.
C
~ytes.',',~The immunogenicity of tumor cells may be enhancedby GM-CSF through facilitating "tumor antigen"
presentation.8 GM-CSF has also been shown to cooperate
with other cytokines in the expansion of specific T cells?
Transfection of the GM-CSF gene to tumor cells with the
aim to increase the immunogenicity of the cells and to locally
stimulate effector cells might also bean interesting approach."
GM-CSF alone might induce tumor regression in humans,
probably by activating specific and unspecific killer cells.",'*
Therapeutic unconjugated monoclonal antibodies (MoAbs)
may eradicate tumor cells in vivo by activating various immune f~ncti0ns.l~
Some of these mechanisms such as antibody-dependent cellular cytolysis and induction and amplification of an idiotypic network response may be augmented
by GM-CSF.14"6Based on our therapeutic experience with
the anticolon carcinoma MoAb 17-1A (mouse IgG2A)
alone,I5 on in vitro results combining GM-CSF and MoAb
17-1A for killing of human carcinoma cell lines14 and on
the above-mentioned effects of GM-CSF, a therapeutic trial
in patients with metastatic colorectal carcinoma (CRC) was
initiated combining GM-CSF and MoAb 17-1A. Long-lasting complete remissions were noted.17 The effects on the
cytotoxic capability of peripheral blood mononuclear cells
in vivo and on cell subsets involved in the cytotoxic reactions
have recently been described."
Detailed information on various aspects of exogenously
administered GM-CSF should be of value to optimize the
use of this cytokine as an antineoplastic and hematopoetic
stimulatory agent. In this report, we describe the pharmacokinetics of GM-CSF and the induction of anti-GM-CSF
antibodies as well as some clinical effects connected to the
induction of these antibodies. The antibody response was
also related to whether the patients were nonimmune-compromised (no concomitant chemotherapy) or immunocompromised (concomitant intensive chemotherapy).
OLONY-STIMULATING FACTORS (CSFs) have
pleiotropic effects, inducing maturation and functional
activation of blood cells. Several hematopoetic growth factors (ie, interleukin-l [IL-l], IL-3, IL-6, IL-11, erythropoetin, stem cell factor, macrophage-CSF, granulocyte-CSF, and
granulocyte-macrophage-CSF [GM-CSF]) have been identified' with the major clinical applications to support myelogeneration after myelosuppressive therapy.
GM-CSF might also be a useful cytokine for biotherapy
of malignant diseases. GM-CSF induces (1) differentiation
of monocytes to large macrophage-like cells?3 ( 2 ) augmentation of major histocompatibility complex class 11 antigen
expression on monocyte^?^ (3) enhancement in vitro of
macrophage and granulocyte spontaneous cytotoxicity and
antibody-dependent cellular cytolysis,6 and(4) increased expression of adhesion molecules on granulocytes and mono-
From the Department of Oncology (Radiumhemmet) and the Immunological Research Laboratory, Karolinska Hospital, Stockholm,
Sweden; Behringwerke AG, Marburg, Germany; and the Research
Department, Karolinska Pharmacy, Stockholm, Sweden.
Submitted August 23, I993; accepted August 25, 1994.
Supported by grants from the Swedish Cancer Society, the Cancer
Society in Stockholm, theKing Gustaf V Jubilee Fund,theElina
Andersen Foundation, theEva and Jerzy Cederbaum Foundation,
and the Karolinska Institute Foundations. The study was approved
by the Ethics Committee of the Karolinska Institute.
Address reprint requests to HikanMellstedt, MD, PhD, Associate
Professor, Department of Oncology (Radiumhemmet), Karolinska
Hospital, S - l 71 76 Stockholm, Sweden.
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.
0 1994 by The American Society of Hematology.
0006-497I/94/84I2-06$3.00/0
4078
Blood, Vol 84, No 12 (December 15). 1994 pp 4078-4087
F
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4079
ANTI-GM-CSFANTIBODIES-BIOLOGICEFFECTS
Table 1. Characteristics of Patients With CRC
Age
(YrVSex
Primary
Tumor Site
(Tumor
Differentiation)
MACC
Previous
Therapy
PS-T
of
(MO)
1
2
3
53ff
63F
661F
Colon (M)
Rectum (M)
Colon (M)
c3
B2
D
S
S
S
2
115
17
4
5
6
651M
671F
291M
Rectum (M)
Colon (M)
Colon (P)
D
c3
Chemo
S
S
S
14
1
3
7
14/F
Colon (M)
c3
S
Chemo
lrrad
9
8
9
10
11
12
13
14
15
16
17
18
19
20
70m
51m
69m
23m
66m
45m
38ff
69/M
78/M
661M
53lM
74F
29/F
Colon (P)
Rectum (M)
Colon (M)
Rectum (M)
Rectum (M)
Colon (“P)
Colon (M)
Colon (M)
Rectum (M)
Rectum (M)
Colon (M)
Rectum (M)
Colon (M)
B3
D
D
c3
B3
c2
c3
B3
B1
c3
c2
c3
D
S
S
3
3
3
9
48
2
2
6
59
2
22
29
S
4
Patient
No.
D
S
S
S
S
S
S
S
S
S
S
No. of
GM-CSF
Treatment
Cycles
Total
dose of
GM-CSF
(pg)
Mesenteric nodes
Left cervical nodes
Lung
3
4
3
9,140
10,500
13,500
1.933
0.500
1.686
66
38
66
Liver
Liver
Liver, Lung, Abdominal
mass
Liver, lymph nodes
3
4
2
15,000
16,000
8,000
1.009
1.732
0.503
66
94
38
3
13,000
1.595
66
Retroperitoneal wall
Liver
Liver
Omentum
Abdominal wall
Lymph nodes
Lymph nodes
Abdominal wall
Liver
Lymph nodes
Liver, Abdominal wall
Pulm
Liver, Lymph nodes
4
3
4
4
4
4
4
4
2
4
4
4
4
17,400
13,200
19,000
18,800
19,000
19,000
16,000
2 1,600
9,200
19,600
18,300
17,600
16,200
2.069
1.282
1 .g70
1.739
2.085
0.430
1.966
0.350
0.600
<0.300
1.155
0.981
0.430
66
66
94
94
66
94
66
85
57
Site
Metastases
Maximum
Anti-GM-CSF
(IgG) Titer
(OD)
Day of Maximum
Antibody Titer
From Start of
Therapy
94
94
85
Abbreviations: M, moderately differentiated; P, poorly differentiated; MACC, modified Astler Coller class; S, surgery; Chemo, chemotherapy;
Irrad, irradiation; PS-T, time from primarysurgery to start of therapy.
Treatment Schedule
MATERIALS AND METHODS
Patients
CRC. Twenty patients (12 males and 8 females) entered a phase
II trial (Table l). The median age was 65 years (range, 14 to 78
years). All patients had metastatic CRC and a Karnofsky index of
280%. One patient had received chemotherapy and irradiation 5
months earlier, and another had chemotherapy 2 months previously.
All others were, except for primary surgery, untreated.
Multiple myeloma (MM). Eight patients (2 menand 6 women)
with MM clinical stage IIA or IIIA according to Durie and Salmon”
were. included (Table 2). The diagnostic criteria for MM have been
described previously.2”The median age was 69 years (range, 50 to
74 years). All patients were previously untreated and had a Karnofsky index of 280%.
CRC patients. Recombinant human GM-CSF (rhGM-CSF) produced in Escherichia coli (specific activity, 5 X lo7 Ulmg protein;
Behringwerke AG, Marburg, Germany) was administered at a dose
of 250 ,ug/m2/d subcutaneously (SC) for 10 days. On day 3,400 mg
of MoAb 17-1A (mouse IgG,; Centocor, Malvern, PA) was infused
intravenously (IV) for 60 minutes. The treatment cycles were repeated every fourth week. Four cycles were administered.” If the
disease progressed during the study period, the patients were withdrawn from the study and were offered chemotherapy.
MM patients. The MM patients entered a phase I multicenter
trial studying escalating doses of cyclophosphamide in combination
with interferon-a (IFN-a) and betamethasone. The dose of cyclophosphamide (Cyclofosfamid; Orion, Espoo, Finland) ranged from
Table 2. Characteristics of Patients Wkh MM
Patient No.
1
2
3
4
5
6
7
8
Clinical Age
(Yr)/Sex
3
72lM
2 4F
681F
4
71F
3
6
5
69lF
4
67lM
Stage
111
<0.300
A
II A
<0.300
111 <0.300
A
II A
II A
II A
111 A
II0.312
A
No. of GM-CSF
Treatment Cycles
8,600
8,600
18,400
16,000
14,800
415,200
Total Dose of
Cyclophosphamide
(mg)
10,200
6,800
12,400
4,000
<0.300
14,400<0.300
12,400
9,270
7,600
Total Dose of
GM-CSF ( p g )
Maximum
Anti-GM-CSF
(IgG) Titer (OD)
13,200
2 1,600
<0.300
~0.300
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RAGNHAMMAR ET AL
4080
500 mg/m2 to 950 mg/m* IV. CY was administered on days 1 and
3 of a 4-day treatment cycle. IFN-(Y(WellferonR; Wellcome Ltd,
Beckenham, London, UK) was administered daily SC on days 1
through 4. Betamethasone (30 mg daily; BetapredR; Glaxo, Middlesex, UK) was administered orally on days 1. through 4. The cycle
was repeated every fourth week. All MM patients received 5 pg/
kgldof
GM-CSF (molgramostim; E coli-derived; LeucomaxR;
Schering-Plough Corp, Kenilworth, NJ) SC from day 5 of a cycle
until the day when the granulocyte count exceeded 1.5 X IO’IL
and the nadir platelet count was passed. The individual number of
treatment cycles as well as the total dose of rhGM-CSF are shown
in Table 2. In all MM patients, GM-CSF was administered for 7 to
12 consecutive days, with a median time of 9 days.
Serum samples for anti-GM-CSF antibody analyses were drawn
at least 10 days after a GM-CSF injection.
Blood Cell Counts
The total number of white blood cells (WBCs) were counted using
a Zeiss standard RA microscope in ordinary light (magnification X
1,000). Two hundred cells were counted. The percentage of WBC
subsets was determined by a differential count on smears of peripheral blood stained with May-Griinwald and Giemsa. The total number of blood cell populations was calculated from these figures.
Serum Sampling
Venous blood was collected in sterile tubes. The serum was separated and stored at -70°C until assayed.
GM-CSF Assay
The pharmacokinetics of GM-CSF were determined on the first
day of a cycle. GM-CSF was assayed in enzyme-linked immunosorbent assay (ELISA), using flat-bottomed microtiter ELISA plates (96
wells; Costar, Cambridge, MA), coated with a mouse monoclonal
antihuman GM-CSF antibody (IgG,; Genzyme, Cambridge, MA,
USA) overnight at 4°C. After blocking with 8% boiled fat-free milk
(Semper, Stockholm, Sweden) diluted in phosphate buffer (pH 9.7)
for 2 hours at 37“C, the serum samples and the GM-CSF standard
(in human normal AB serum [1:6]; Behringwerke AG) diluted in
phosphate buffer (pH 7.2) were added in duplicates and incubated
overnight at 4°C. The serum samples as well as the GM-CSF standard were passed through a Protein A Sephadex column (CL-4b;
Pharmacia, Uppsala, Sweden) before adding to the ELISA plates to
remove cross-reacting antibodies.21Then, a polyclonal rabbit antiGM-CSF antibody (Genzyme) in phosphate buffer, pH 7.4, (1:lOOO)
containing 1% milk was added for 2 hours at 37°C. The plates were
then reacted for 2 hours at 37°C with a goat antirabbit IgG alkaline
phosphate conjugate (Sigma, St Louis, MO) in phosphate buffer, pH
7.4, with 1% milk (1:300). After enzyme reaction for 30 minutes at
room temperature using p-nitrophenylphosphate (1 mg/mL; Sigma)
in diethanolamine buffer, pH 9.8, the absorbance was read at 405
nm using an automatic ELISA reader (Multi-Scan plus; Laboratory
Systems, Helsinki, Finland).
Anti-GM-CSF Antibody Assay
Antibodies to rhGM-CSF were detected by a solid-phase, indirect
ELISA. Briefly, round-bottom microtiter plates (Behring Diagnostics, Marburg, Germany) were coated with 125 MLper well of rhGMCSF (1 .O pg/mL) in 50 mmoVL phosphate buffer, pH 7.2, at room
temperature overnight. (The rhGM-CSFBehringwerke preparation
used to coat the plates was shown by mock-ELISA to contain 2.8
ppm of E coli-derived contaminants.) Serum samples were diluted
1 :20 in a TRIS/HCL-buffer, pH 8.1, containing lactofemn, gelatine,
bovine serum, and 0.5% Tween 20 (product no. OWBE; Behringwerke AG) for detection of IgG antibodies. To determine antiGM-CSF IgM antibodies, the sample was further diluted ( I : l [vol/
vol]) with rheumatic factor (RF) adsorbent (product no. OUCG 14:
Behringwerke AG) and incubated at room temperature for 15 minutes. (The RF adsorbent binds RF, which might give false-positive
results in an IgM antibody assay.) The serum sample was then mixed
with an E coli lysate (2%; prepared from the E coli strain LE 392
K12 USS; Behringwerke AG) and incubated at room temperature
for 1 hour. (This procedure was introduced to remove E cdi-directed
antibodies.) A total of 100 pL of the serum samples was then added
to microtiter wells in duplicate and incubated at room temperature
for 2 hours. The wells were washed in phosphate-buffered saline
containing 0.5% Tween 20. The plates were then incubated with 0. I
mL per well of peroxidase conjugated antihuman IgM ( I :40; product
no. OSDJ; Behringwerke AG) or antihuman IgG ( 1:40; product no.
OSDH; Behringwerke AG), respectively, for 2 hours at room temperature. After washing 4 times, 0.1 mL of the substrate tetramethylbenzidine (product no. OUVOlO; Behring Diagnostica) was added
to each well, and the plate was incubated for 30 minutes at room
temperature. The enzyme reaction was stopped by adding 0.5 N
sulfuric acid. The plate was read at 450 nm using a Behring ELISA
Processor BEP I1 (Behring Diagnostica).
Serum samples with an optical density (OD) value above the range
of 300 normal blood donors were considered positive, were further
quantitated by ELISA, and were applied to Western blot analysis as
a confirmatory assay. The OD values were related to a synthetic
standard composed of rabbit antihuman-GM-CSF Fab’ fragments
chemically linked to human IgG or IgM molecules, respectively
(patent no. EPA 291086 [Boehringer Mannheim] and DEOS
31 12334 [Behringwerke AG]). The synthetic standard has the property of binding to GM-CSF by the Fab’ fragment of rabbit Ig and to
antihuman Ig antibodies through the human Ig part of the molecule.
Western Blotting
rhGM-CSF was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 16%) using the Laemmli
buffer system.22The protein was electroblotted into nitrocellulose
filters (Hybond C-Extra; Amersham, Braunschweig, Germany) according to the method of Towbin et aIz3using 25 mmol/L Tris to 192
mmol/L glycine/20% methanol buffer (pH 8.3; vol/vol). A voltage
gradient of 6 V/cm was applied for I hour. Each blot was checked
by AuroDye protein staining (Amersham). After blotting, the filter
was immersed in blocking solution (TRIS/HCI buffer, pH 8.l), containing lactofemn, gelatine, bovine serum, and0.5% Tween 20
(product no. OWBE) and agitated for 1 hour at room temperature.
The samples (diluted 150 in OWBE) were preincubated without
or with E coli lysate (2%) for 1 hour at room temperature. The filter
was then incubated with the samples at room temperature overnight
under continuous agitation. After 5 washes in phosphate-buffered
saline containing 0.5% Tween 20, the filter was incubated for 1 hour
at room temperature with a goat antihuman IgG labeled with colloid
gold (Amersham). The signal was amplified, according to the manufacturer’s instructions, using a silver enhancement kit (IntenSE; Amersham). In each test, a positive serum was included showing the
same staining pattern as the synthetic standard.
Serum Immune Complexes
Immune complexes in serum were analyzed using the C l q binding
technique as described in detail previo~sly.’~
Pharmacokinetics
The pharmacokinetics parameters for subcutaneous administration
were evaluated and fitted by a one-compartment model with the
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ANTI-GM-CSF ANTIBODIES-BIOLOGIC
EFFECTS
4081
5r
Time (hour)
Fig 1. Serum concentration (mean) of GM-CSF after a single SC
injection (250 pg/m*) at treatment
cycle I (0;n = 6; CRC patients no.
10, 11, 12, 14, 18,
and 19) and treatment cycles 111 or IV (0;n = 5;
CRC patients no. 11, 12, 14, 18, and 19). respectively. (All 5 patients
analyzed at cycles 111 or IV had anti-GM-CSF lgG antibodies]. The
difference in AUC was statistically significant ( P c .005).
first order of absorption. Parameter estimation using nonlinear least
squares analysis was performed using PCNONLIN (Statistical Consultants Lexington, KY).
Statistical Analyses
The differences between means were analyzed using Student's rtest or Wilcoxon signed rank test for paired and unpaired observations. The linear regression model was used to estimate correlation
between independent observations.
RESULTS
Pharmacokinetics of GM-CSF
GM-CSF wasnot detectable in serum before therapy.
After an SC injection of 250 pg/m' of GM-CSF at cycle I,
the maximum calculated concentration (C,,,=) was 5.24 -+
0.56 ng/mL and was reached at 3.25 2 0.25 hours after the
injection. The C, at cycles I11 and IV in the same patients
was 2.87 -+ 0.57 ng/mL after 2.67 2 0.33 hours. The difference in serum C,, was statistically significant ( P < .05).
The corresponding serum half-lives (TI,') were 2.91 -+ 0.8
hours and 1.59 -+ 0.2 hours, respectively ( P < .OS). There
was also a statistically significant difference in the area under
the concentration curve (AUC) comparing cycle I (30.86 2
6.03 hours X ng/mL) with cycles 111 and IV (14.90 2 4.10
hours X ng/mL; P < ,005; see Fig 1). The decrease in C,,
and AUC by increasing number of treatment cycles was
related to the induction of GM-CSF antibodies (see below).
Induction of Anti-GM-CSF Antibodies
results, the serum samples were preincubated with an E coli
lysate to adsorb anti-E coli antibodies. Analyses of serum
samples not preincubated with the E coli lysate gave higher
OD values (data not shown). Therefore, our assay system
has an improved specificity for anti-GM-CSF antibodies.
An OD value of greater than 0.3 was used as cutoff for
anti-GM-CSF IgG antibodies, and an OD value of greater
than 0.15 was usedfor IgM antibodies. In the control population of 300 healthy blood donors, greater than 95% of the
individuals had OD values below these levels. Of the 300
donors, 5 had antibodies against GM-CSF detected by
ELISA, which wasconfirmed by a weak but specific staining
in Western blot (data not shown).
No CRC patients had antibodies against GM-CSF before
therapy. Anti-GM-CSF IgM antibodies were not detected
with the present sampling design. During GM-CSF therapy
in the CRC patients, increasing titers of specific anti-GMCSF IgG antibodies were noted (Figs 2 and 3 and Table l).
The GM-CSF antibody titers had disappeared at about 30
weeks after the last injection (Fig 2). At the end of cycles
I1 and IV, 50% and 90%, respectively, of the tested patients
had IgG antibodies (Fig 3). In total, 19 of the 20 CRC
patients (95%) developed anti-GM-CSF IgG antibodies (Table l). Three groups of CRC patients could be distinguished
with regard to the level of antibody titers induced: no antibody titer (n = 1; CRC patient no. 17); low antibody titers
(n = 5; CRC patients no. 2, 6, 13, 15, and 20); andhigh
antibody titers (n = 14; CRC patients no. 1, 3, 4, 5, 7, 8, 9,
10, 11, 12, 14, 16, 18, and 19).
The presence of anti-GM-CSF IgG antibodies was confirmed by Western blot analyses in all patients, as shown in
Fig 4. In addition to the GM-CSF bands (14.5 kD), several
weak bands with a molecular weight (MW) corresponding
to the MW of E coli proteins (ca 20 kD) could be observed
after protein staining (Fig 4, lane A). Immunoblotting of sera
preincubated with E coli lysate using an antihuman IgG
antiserum showed no bands at the place of E coli proteins
but showed only antibodies reacting with GM-CSF (Fig 4,
lane B). Sera not preincubated with E coli lysate showed the
]A
TI
c
c
,
,
TI1
TV
c,
L)
A high proportion of normal individuals has antibodies
against E coli, usually of high affinit~.'~Such antibodies
may interfere in the assay system because the rhGM-CSF
preparation for coating of the plates might contain minute
amounts (2.8 ppm) of E coli proteins. To avoid false-positive
E
2P
13
""-
"""""-"""""""""
"""
ao
l2
28
Jx
Time (weeks)
Fig 2. Anti-GM-CSF IgG antibody titers (ELISA; mean) of patients
In = 19) treated with SC injections of GM-CSF for 10 days every
month for 4 months. Arrows show treatment cycles; (---l indicates
cutoff level.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
RAGNHAMMAR ET AL
4082
I
10
(n.20)
IO
I
I
II
I
(n.20)
IO
I I,
1
111
IV
(nzls)
(11.13)
Fig 3. Frequency of patients with anti-GM-CSF lgG antibodies at
day 1 and day 10 of each treatment cycle (Ithrough IV).Patients with
no detectable anti-GM-CSF antibodies (m) and with anti-GM-CSF
of patients
antibodies (W). Figures with brackets indicate the number
receiving therespective GM-CSF cycle.
presence of antibodies against E coli as well as GM-CSF
(Fig 4, lane C). Blotting of the synthetic antibody standards
(IgG and IgM) showed a similar pattern to that of the induced
antibodies (Fig 4, lanes D and E). In 2% of the 300 normal
donors, anti-E coli IgG antibodies could be detected (Fig 4,
lane F) that disappeared after preincubation with E coli lysate
(Fig 4, lane G).
From an immunologic point of view, the CRC patients
might be regarded as having a noncompromised immune
systems because they had not previously received chemotherapy (with the exception for 2 patients who had been adminis-
A
'1)
21)
41)
51)
B
C
D
E
tered cytostatics 2 to 5 months earlier). All CRC patients had
a good performance status. For comparison, a group ofMM
patients who had been treated with an intensive chemotherapy
regimen followed by GM-CSF administration to support bone
marrow regeneration (immunocompromised patients) was included. The MMpatientsreceived
rhGM-CSF in a similar
scheduling as the CRC patients (see Materials and Methods).
Thenumber of cyclesadministeredbeforetestingforantiGM-CSF antibodies in MM patients varied between 2 and 6
(median no., 4). Only 1 MM patient (MM patient no. 8) had
detectable anti-GM-CSF antibodies after
4 GM-CSF treatment
cycles. The antibody titer was very low. The
OD value (0.312;
see Table 2 and Fig 5 ) was just above the cutoff level(0.300),
and a weak band was noted on Western blot (data not shown).
In the remaining 7 patients, no anti-GM-CSF antibodies were
detected by ELISA and Western blot (Fig 5). The difference
in number of patients with regard to anti-GM-CSF antibody
induction between the two groups was statistically, highly significant (x' = 5.23; P < .001).
The two patient groups both received E coli-derived GMCSF. However, CRC patients were administered a recombinant protein produced by Behringwerke, and MM patients
received rhGM-CSF from Schering-Plough. As can be observed in Fig 5, CRC patients treated with rhGM-CSFIBehringwerke developed antibodies reacting withrhGM-CSF/
Behringwerke as well as with rhGM-CSF/Schering-Plough.
Clinical Effects of Anti-GM-CSF Antibodies
Pharmcrcokinetics. The induction of anti-GM-CSF antibodies was associated with a significant reduction of C,,,
TI,?.and AUC of exogenously administered GM-CSF (see
above and Fig 1).
F G
Fig 4. Western blotting of anti-GM-CSF antibodies (CRC patient no. 9). Mobilities of the MW standards are indicated at the margin:
left
(1) bovine trypsin inhibitor, 6.2 kD; (2) lysozyme, 14.3 kD; (3) plactoglobulin, 18.4 kD; (4) carbonic anhydrase, 28.5
kD; and (5) ovalbumin, 43.8 kD. (Horizontal arrow
indicates the localizationof the E coli bands.) Lane
A shows AuroDye staining of blots. Note the GMCSF main bandwith an MW of 14.5 kD and the main
€ coli bands at ca. 20 kD. LaneB shows immunoblotting of a patient serum after preincubation with E
coli lysate. Note only visible bands at the place of
GM-CSF. LaneC is the
same as for lane B but without
preincubation with € coli lysate. Note also the presence of anti-€ coli antibodies. Lanes D and E show
immunoblotting after adding the anti-GM-CSF IgM
(D) and anti-GM-CSF IgG (E) synthetic standards.
Lane F shows immunoblottingof a normal blooddonor serum without preincubation with€ coli lysate.
Note the detectionof anti-€ coli antibodies. Lane G
is thesame as for lane F but withpreincubation with
E coli lysate. Note the disappearance of anti-€ coli
antibodies.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
4083
ANTI-GM-CSFANTIBODIES-BIOLOGICEFFECTS
4-
n
3-
Fig 5. Anti-GM-CSF IgG antibody titers IEUSA)
in 10 CRC patients ( s e e Table 1) and 8 MM patients
(see Table 2) tested against plates coated with GMCSFIBehringwerke).( or GM-CSFISchering-Plough
(0).
titer and total number of WBCs ( r = -0.438; P < .005),
neutrophils ( r = -0.467; P < .001), and eosinophils ( r =
-0.334; P < .05) when comparing day 10 of all treatment
cycles. By increasing anti-GM-CSF titers, the cell counts
decreased. In patients with lowantibody titers, no such correIation could be noted.
After each treatment cycle, WBC counts returned to baseline level within 5 days. There was no significant influence
on the total number of WBCs during long-term follow-up
(Fig 7).
Side effects. The main side effects in all CRC patients
at cycle I that were considered to depend on the GM-CSF
treatment were myalgia (50%), local irritation (45%), fever
(greater than38.5"C; 35%), bone painskternalgia (35%),
rashlpruritus (35%), conjunctivitis (30%), headache (25%),
nausea (15%), and chills (15%). Myalgia was easily controlled by dextropropoxiphene. However, in 1 patient (CRC
pateint no. l), the GM-CSF dose had to be reduced because
of severe myalgia. Myalgia appeared at days 2 to 3 and
disappeared at days 8 to 9 of the cycle. Fever (greater than
WBCs. At treatment cycle IV, in CRC patients, the maximum total numbers of WBCs, neutrophils, and eosinophils
were significantly lower than the peak values at the previous
cycles (P < .05). There was also a clear tendency by each
treatment cycle for gradually decreased monocyte counts at
day 10 ( P = .06). No tendency for a decreased increment
of lymphocytes was noted (Table 3). Patients with low antiGM-CSF antibody titers showed significantly higher values
of WBCs, eosinophils, and neutrophils at day 10 of cycle IV
as compared with patients with high anti-GM-CSF antibody
titers (Table 4). The individual WBC counts at day 10 of
each cycle for those 13 patients completing 4 treatment
courses are shown in Fig 6. The total number of WBCs
was unchanged during the 4 treatment cycles in the patient
(CRC17) not developing anti-GM-CSF antibodies, whereas
there was a modest decrease in the low-titer patients and a
marked decrease at treatment cycles 111 and/or IV in the
high-titer group.
Moreover, in the high-titer group, there was a statistically
significant correlation between the anti-GM-CSF antibody
Table 3. Total No. of Blood Cells (Mean f: SE; x lo-' L) at Days 1 and 10 of the 4 Treatment Cycles
Cycle l
(n = 20)
Blood Cells
WBC
Neutrophils
Eosinophils
Basophils
Monocytes
Lymphocytes
t
5
Cycle II
(n = 20)
Day 1
Day 10
6.66 2 0.35
4.42 t 0.35
0.27 2 0.05
0.02 2 0.01
0.42 2 0.05
1.53 2 0.10
26.24 2 1.78'
17.12 2 1.58t
4.47 2 0.57t
0.06 f 0.02
1.16 2 0.23t
2.72 f 0.24'
Day 1
6.35 2
4.21 2
0.21 2
0.02 2
0.37 +1.54 +-
0.39
0.37
0.03
0.01
0.05
0.12
Cycle IV
In = 13)
Cycle 111
(n = 18)
Day 10
30.41 2
19.24 2
6.38 2
0.04 2
0.96 2
3.27 2
1.97*
1.31t
0.79t
0.02
0.17t
0.32'
P < .05 and " P < ,001 comparing day 1 and 10 of each cycle.
P < .05 and * P < .01 comparing day 10 of cycle I and day 10 of cycle IV.
Day 1
Day 10
Day 1
Day 10
6.54 ir 0.40
4.27 2 0.43
0.32 2 0.06
0.03 t 0.01
0.44 2 0.08
1.51 2 0.17
22.65 2 3.81"
13.97 t 2.53"
4.41 +- 1.05'
0.04 t 0.02
0.85 t 0.13t
2.80 -e 0.33"
7.34 2 0.56
5.15 +- 0.69
0.33 +- 0.13
0.04 ? 0.02
0.43 t 0.04
1.53 +- 0.18
15.83 2 2.48+*
9.65 +- 1.50tS
2.68 -c 0.69t5
0.02 5 0.01
0.80 0.11t
2.57 t 0.45t
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
RAGNHAMMAR ET AL
4084
Table 4. Total No. of Blood Cells (Mean 2 SE; x lo-’ L) in CRC Patients With Low (n = 4) and High (n = 8 ) Anti-GM-CSF Antibody Titers,
Respectively, at Days l and 10 of Treatment Cycle IV
Day 1
Low Anti-GM-CSF
Blood Subsets
WBC
Neutrophils
Eosinophils
Basophils
Monocytes
Lymphocytes
Antibody Titer
6.63
4.34
0.19
0.04
0.52
1.92
i- 0.84
2 1.07
i 0.09
t 0.01
2 0.14
i 0.36
Day l 0
High Anti-GM-CSF
Antibody Titer
7.72
5.40
0.37
0.04
0.40
1.41
i 0.71
0.84
i 0.16
t 0.01
i 0.04
i 0.20
-t
Low Anti-GM-CSF
Antibody Titer
High Anti-GM-CSF
Antibody Titer
23.43 i 3.64
13.95 -t 1.38
4.65 i 1.46
0.02 -t 0.01
0.99 2 0.08
3.89 -t 1.12
: 2.34*
12.04 :
7.50 z 1.71*
1.70 2 0.49*
0.03 -t 0.01
0.70 ir 0.16
3.69 t 0.91
Only patients with low (CRC patients no. 2,13,15, 20) and high (CRC patients no. 5, 8, 10, 11, 12, 14, 18,
4 GM-CSF treatment cycles are depicted in this table.
* P < .05 comparing the low- and high-titer groups at the same dayof the treatment cycle.
38.5”C) was noted 2 to 3 hours after the injection and lasted
for 2 to 6 hours. There was a clear tendency towards reduced
frequency of systemic adverse reactions such as myalgia,
fever, rusWpruritus, conjunctivitis, headache, and nausea by
treatment cycle IV. The side effects during each cycle in
those 13 CRC patients completing 4 GM-CSF treatment
courses are shown in Table 5. Furthermore, in the high antiGM-CSF antibody titer group, a total of 4 systemic adverse
events were registered among 9 patients at treatment cycle
IV, whereas 7 were recorded among the 4 low antibody titer
patients.
There were no clinical signs of immune complex-related
adverse reactions. The kidneys are supposed to be the most
sensitive organ to immune complexes. No patient had proteinuria or red blood cells in the urine, which are sensitive
indicators of renal immune complexes depositions. Serum
immune complexes were analyzed at each treatment cycle.
No circulating immune complexes could be detected.
DISCUSSION
Nonimmunocompromised patients with metastatic CRC
received 10 days of GM-CSF treatment repeated every
month for 4 months with the aim being to activate cellular
cytotoxic functions as well as antigen presenting cells and
to amplify an idiotypic immune network response. The pharmacokinetics (C,,,, TI,>,and AUC) of GM-CSF during cycle
I was similar to that described by other^.^^^^' However, after
repeated cycles, pharmacokinetics analyses of GM-CSF
showed significantly reduced C,,,,,
andAUCthat correlated with the appearance of anti-GM-CSF antibodies.
To acertain that our assay system detected true anti-GMCSF antibodies, particular attention waspaidto avoiding
measuring anti-E coli antibodies, because minute amounts
of E coli proteins might be present in the drug preparations
administered to the patients aswell as in the rhGM-CSF
preparations used to capture anti-GM-CSF antibodies. Morover, anti-E coli antibodies are common among normal individuals and increase with an individual’s age.25By preincubation of the sera with an E coli, lysate false-positive results
could be avoided. These precautions should be taken in all
immune assays using recombinant proteins producedin E
19) anti-GM-CSF titers completing
coli. Furthermore, human antimouse antibodies (HAMAs)
were induced in all CRC patients, but HAMAs did not seem
to interact with anti-GM-CSF antibodies in the assay. The
incubation medium contained whole bovine serum that effectively suppressed nonspecific binding to the solid phase.
Nonbound HAMAs were washed away before adding the
conjugate. Moreover, there was no correlation between the
HAMA response and the anti-GM-CSF response. The induction of HAMAs was more rapid than that of anti-GMCSF antibodies. Most of the CRC patients had developed
HAMAs at start of cycle 11, whereas no patient had antiGM-CSF antibodies at this time-point. At 30 weeks after
completion of therapy, no patient had detectable anti-GMCSF titer, whereas all patients still had HAMAs (submitted
for publication).
The induction of anti-GM-CSF antibodies was probably
a primary immune response even though no IgM antibodies
were detected. The kinetics of the IgG antibodies were
clearly that of a primary immunization. Reports on induction
of anti-GM-CSF antibodies are scanty. Of 16 patients with
advanced malignancies who were heavily pretreated or on
intensive chemotherapy, 4 developed anti-GM-CSF IgG antibodies against rhGM-CSF.” Of 16 patients with myelodysplastic syndrome receiving GM-CSF after previous treatment with chemotherapy, 1 developed a low titer of antiGM-CSF antibodiesz9 In spite of an abundance of studies
using rhGM-CSF to support myeloregeneration after myelosuppressive therapy, to our knowledge, these are the only
studies that show anti-GM-CSF antibody induction. These
two reports are in agreement with the results in the MM
patients of the present study, where l of 8 patients on heavy
chemotherapy had a low anti-GM-CSF antibody titer after
4 cycles of GM-CSF administration.
The high frequency of anti-GM-CSF antibodies inthe
CRC patients may have several explanations. All patients
had a good performance status, and only 2 of them had
previously received chemotherapy/irradiation.Therefore, the
CRC patients should be regarded as nonimmunocompromised as compared with the MM patients who, like the patients of the other studies, were most likely immunocompromised as a consequence of intensive chemotherapy.
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
4085
ANTI-GM-CSFANTIBODIES-BIOLOGICEFFECTS
40
30
20
10
20
30
40
50
Time (weeks)
Fig 7. Total WBC counts (mean) during and after GM-CSF treatment. Arrows indicate treatment cycles.
10
0
I
I
I
II
111
I
IV
Treatment cycle
Fig 6. Total number of WBCs at day 10 of the 4 GM-CSF treatment
cycles in those 13 patients completing 4 cycles (Table 1) is shown in
relation to anti-GM-CSF antibody titers. (.I no anti-GM-CSF antibodies (CRC patient no. 171; (@) low anti-GM-CSF antibody titers
(CRC patients no. 2, 13, 15, and 20); (A)high anti-GM-CSF antibody
titers (CRC patients no. 5, 8, 10, 11, 12, 14, 18, and 19).
Therefore, our results might support the assumption that chemotherapy prevented the development of antibodies to
rhGM-CSF. Moreover, we administered GM-CSF SC, which
is an excellent route for antigen presentation as compared
with IV administration. Furthermore, GM-CSF was administered in repeated cycles that boostered the patients.
Interestingly, antibodies developed against GM-CSF/
Behringwerke also reacted with GM-CSF/Schering-Plough.
(Both GM-CSFs were produced in E coli.) The standards as
well as most of the positive serum samples showed higher
signals on GM-CSF/Schering-Plough-coated plates as compared with GM-CSFBehringwerke-coated plates (Fig 5).
This might partly be because of the fact that, in this set-up
of analyses, freshly coated GM-CSFISchering-Ploughplates
were used, whereas the GM-CSFBehringwerke plates were
precoated and stored with a drying agent in sealed bags at
+4"C until use. However, there was no constant factor from
sample to sample between plates coated with the different
GM-CSFs. This is not surprising. The two GM-CSFs were
prepared by different production procedures. The epitope
pattern of the two different GM-CSFs would be expected to
differ, and the antibody response may vary from patient to
patient with regard to epitopes recognized.
The presence of GM-CSF antibodies seemed to have biologic effects. The altered pharmacokinetics of GM-CSF with
decreased C, and AUC as well as shorter T I n resulted
probably in reduced amounts of bioavailable rhGM-CSF,
which was also suggested by Gribben et al.'' In that study,
the increase in neutrophils was not that expected in 1 of 4
patients developing anti-GM-CSF antibodies. In our patients, there was a clear relationship between the anti-GMCSF antibody titers and the increase in WBCs, neutrophils,
and eosinophils (ie, the higher the antibody titer, the lower
the cell increase). With repeated cycles, there was a clear
tendency towards a lower increment in total number of
monocytes, although the difference did not reach statistical
significance. Lymphocyte counts seemed to be uneffected.
The results are in agreement with the notion that GM-CSF
mainly affects maturatiodproliferation of hematopoetic cells
Table 5. Frequency of GM-CSF Side Effects in Those 13 Patients
Completing 4 GM-CSF Treatment Cycles
Treatment Cycle
Side Effects
Myalgia
Local irritation
Fever >38.5"C
Bone pains/sternalgia
Rashlpruritus
Headache
Conjunctivitis
Nausea
Chills
Percentages are shown in parentheses.
II
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4086
RAGNHAMMAR ET AL
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ACKNOWLEDGMENT
15. Mellstedt H, Frodin J-E, Masucci G, Ragnhammar P, FagerDr Jiirgen Frisch is greatly acknowledged for stimulating discusJ, Wersall P, Osterborg A: The therapeutic
berg J, Hjelm A-L, Shetye
sions. We thank Birgitta Hagstrom, Annika Overmo, and Gun Ersuse of monoclonal antibodies in colorectal carcinoma. Semin Oncol
mark for skillful technical assistance; Anita Ljungberg for providing
18:462, 199 I
excellent nursing; and Lena Helgesson and Gunilla Bur& for skill16. Tao M-H, Levy R: Idiotype/granulocyte-macrophage colonyfull typing of the manuscript.
stimulating factor fusion protein as a vaccine for B-cell lymphoma.
Nature 362:755, 1993
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of the myeloid and monocytic lineages. An alternative explanation to the lower leucocyte counts
at subsequent cycles
may bea decreased capacityof bone marrow to bestimulated
by GM-CSF. Moreover, at cycle IV, systemic side effects
of GM-CSF(myalgia,fever,
rash/pruritus,conjunctivitis,
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The induction of anti-rhGM-CSF antibodies did not seem
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antibodies induced against exogenous E coli-derived GMCSF recognized epitopes on the
nativeproteinbackbone,
normallyprotected by 0-linked glycosylationbut exposed
on E coli-produced GM-CSF." Therefore, endogenous GMCSF may not be recognized by these antibodies becausethis
molecule might not express the
relevant immunotopes. AntiGM-CSF antibodies alsogradually disappeared after discontinuation of GM-CSF, probably as aresult of nofurther
antigenic stimulation. Therefore, the effects on endogenous
GM-CSF might not be hampered by
antibodies against exogenous E coli-derived GM-CSF.
In summary, this study showed that repeated SC administrationof E coli-derived GM-CSF in nonimmunocompromised patients induced a primary humoral immune response
against exogenous GM-CSF in practically all patients. The
pharmacokinetics and the variations in WBC counts during
therapy indicated that the biologic effects of the exogenous
GM-CSF werereduced by the induced antibodies. However,
antibodyinductionin
immunocompromisedpatientswas
rare and of low titers. This has to betaken into consideration
when designing therapeutic protocols using recombinant cytokines(probably not only GM-CSF) in patients prone to
mount an immune response.
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