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Biologic Effects of Anti-Interleukin-6 Murine Monoclonal Antibody in
Advanced Multiple Myeloma
By Regis Bataille, Bart Barlogie, Zhao Yang Lu, Jean-Franpois Rossi, Thierry Lavabre-Bertrand, Thad Beck,
John Wijdenes, Jean Brochier, and Bernard Klein
In patients with advanced multiple myeloma (MM) there is
an excess of production of interleukin-6 (IL-6) in vivo, and
elevated serum levelsare associated with plasmablastic proliferative activity and short survival. These data prompted US
t o perform a clinical trial with a murine anti-IL-6 monoclonal
antibody (MoAb) t o neutralize the excess of this putatively
deleterious factor in these patients. Ten MM patients with
extramedullary involvement frequently were treated with
anti-IL-6 MoAb. The MoAb was administered intravenously
t o 9 patients; 1 patient with malignant pleural effusion received intrapleural therapy. Of the 3 patients who succumbed t o progressive MM after less than 1 week of treatment (including the only 1 treated locally), 2 with evaluable
data exhibited marked inhibition of plasmablastic proliieration. Among the 7 patients remaining more homogeneous
receiving the anti-IL-6 MoAb for more than1 week, 3 had
objective antiproliierative effect marked by a significant reduction of the
myeloma cell labellingindex within the bone
marrow. One of these 3 patients achieved a 30% regression
of tumor mass.However, none of the patientsstudied
achieved remission or improved outcome as judged by standard clinical criteria. M major interest, objective antiproliferative effects were associated with complete inhibition of
C-reactive protein (CRP) synthesis and low daily 11-6 production in vivo. On the other hand, the lack of effect in 4 patients
was associated with a higher IL-6 production and inability
of the MoAb t o neutralize it. Anti-IL-6 was also associated
with resolution of low-grade fever in all the patients and
with worsening thrombocytopenia and mild neutropenia.
The generation of human antibodies t o Fc fragment of the
murine anti-IL-6 MoAb observed in 1 patient wasassociated
with dramatic progression. These data show that anti-lL-6
MoAb can suppress the proliierationof myeloma cells and
underscore the biologic role of IL-6 for myeloma growth in
vivo. Furthermore, suppression of CRP and worsening of
neutropenia/thrombocytopenia both indicate that IL-6 is
critically involved in acute-phase responses and granulopoiesidthrombopoiesis.
0 1995 by The American Society ofHematology.
I
standard chemotherapy and with a life expectancy less than 1 month
NTERLEUKIN-6(&6),apleiotropiccytokineproduced
were treated with anti-L-6 MoAb. The clinical course of a tenth
by a range of cells, plays a central role in both host defense
patient treated similarly and previously reported has been included
mechanismsandacute-phaseresponses.’pioneeringstudies
have shown thatthis cytokine controlled the proliferation2” but in the current study, considering new data on L - 6 production in
vivo.I6 Written informed consent was obtained from all the patients.
did not induce thedifferentiation6v7 ofimmature myeloma cells
Selected pretreatment characteristics of the 10 patients are summain vitro. First,anti-L-6 MoAb blocked thespontmwus prolifrized in Tables 1 and 2. All but 1 patient were treated with intraveeration of freshly explanted myeloma
cell in short-term cultures nous (IV) B-E8 anti-IL-6 MoAb, usually at a standard dose of 20
and additionof exogeneous L - 6 further increasedit.s5 Second,
mg/day for at least 4 days (up to68 days). One patient (no. 3)
myeloma celllines whose proliferationis dependent on addition
received B-E8 intrapleurally for a malignant pleural effusion at a
daily dose of 20 mg for 3 days. Response to treatment was evaluated
of exogenwus IL-6havebeenobtained
from everypatient
with extramedullary proliferation?All studies agreed that large using standard criteria, including performance status, body temperaamounts of IL-6 are produced bythe tumoral microenvironment ture, weight, and improvement of symptoms such as anemia, hypercalcemia, Occurrence of infections, and regression of monoclonal
in response to stimulating myeloma ~e&3.3.~.’O
Several studies
component and myeloma cell mass.
have reported an autocrine productionof IL-6 by the myeloma
cells themselves?.” However, this production was only very
Materials and Methods
minor (1/1,OOO) as compared with thatof the tumoral microenInfection ofanti-lL-6 MoAb. Injection of anti-IL-6 MoAb was
vironment. For these reasons, these studies could not exclude
performed in vivo as previously described.16Nine of the 10 patients
a minor contamination by environmental cells or more simply
a weak activation
of L - 6 gene in myeloma cellsby exogeneous
Fromthe Laboratoire Central d’Hdmatologie et Laboratoire
IL-6, as has been previously reported.” The involvement of
d’Oncoginkse Immunohimatologique-lnstitut de Biologie, Nantes,
this cytokine in vivo is supported by the relationship
of inFrance; Arkansas Cancer Research Center, Little Rock, AR; INcreased L - 6 serum levelsto in vivo tumor cell kinetics, disease
SERM U.291, lmmunopathologie des Maladies Tumorales et Autoseverity,andsurvival
ofpatientswith
multiplemyeloma
immunes, Montpellier, France; and Innothkrapie, Besancon, France.
These data led us to develop a clinical trial
to define
Submitted June 16, 1994; accepted February 27, 1995.
the effectsof a murineanti-L-6 monoclonal antibody (MoAb)
Supported by grants from I’Association Pour la Recherche sur le
in patients with advancedMM (mainly plasma cell leukemia).
Cancer, la Ligue Nationale de Lutte contre le Cancer (Paris,
The rationale of this project wasalso supportedby data obtained France), and la Ligue Rigionale de Lutte contre le Cancer (Nantes,
France).
in mice showing that (1) L-6 is a major paracrine plasmacyAddress reprint requests to Rigis Bataille, MD, PhD, Laboratoire
tom growth factor and that(2) anti-L-6 and anti-L6 recepcentral d’Hkmatologie, Institut de Biologie, 9 quai Moncousu, 44035
tor MoAbs significantly inhibitthe in vivo expansion of plasNantes Ckdex 01, France.
macytomas and improve the survival of inoculated mice.I5
PATIENTS, MATERIALS, AND METHODS
Patients
Nine patients with advanced and progressive MM, mainly primary
and secondary plasma cell leukemia (PCL) (n = 6), refractory to
Blood, Vol 86, No 2 (July 15). 1995: pp 685-691
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 1995 by The American Society of Hematology.
0006-4971/95/8602-0011$3.00/0
685
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BATAILLE ET AL
686
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received B-E8 and 1 received mab-8 MoAb, which were administered according to a schedule outlined in Table 1 . Doses of antiIL-6 MoAb were selected to provide serum concentrations similar
to those known to inhibit myeloma cell proliferaion in vitro and
2
were adjusted according to patients' clinical status.
Anti-IL-6 MoAb. Anti-IL-6 MoAb (B-E8 and mab-8, both
IgGK) were prepared by J. Wijdenes (Innotherapie, Besancon,
France) and L. Aarden (Central Laboratory Blood Transfusion Service, Amsterdam, The Netherlands), respectively, by immunizing
mice with rIL-617,1*
These MoAb werepurifiedand submitted to
E
stringent analyses required before use, as previously described.'6
The purified MoAb were sterile and free of pyrogenic activity.
Collection of peripheral blood cells and serum samples. Peripheral blood or bone marrowsamples for all studies, including pharmacokinetic studies, were collected at 7 AM, 1 hour before mab-8 or
B-E8 injections. Peripheral blood serum and plasma were stored at
r
c
-20°C until use. Peripheral blood, pleural effusion, or bone marrow
mononuclear cells were obtained by centrifugation of heparinized
samples over Ficoll-Hypaque gradients.
Proliferation and culture assays of myeloma cells. The percentages of myeloma cells were determined by cytoplasmic immunofluorescence using anti-K or anti-A light chain antibodies directly cou6
C
pledto fluorescein (Kallestadt, Austin, TX). The percentages of
.plasma cells in S phase (ie, the labeling index) were determined
e
._
using an antibromodeoxyuridine MoAb (Immunotech, Marseilles,
U
0
France) and a rhodamine-labeled goat antimouse Ig (Jackson LaboraD
tories, West Grove, PA) ina double fluorescence technique described
c
m
5
elsewhere.' Cells were cultured at lo6 cellslml in RPM1 1640 mem
dium supplemented with 5 X lO-5 m o m 2MEand 5% fetal calf
s
g
serum for 5 days, hence referred to as culture medium. In some
groups, 10 pg/mL of anti-IL-6 MoAb (mab-8 or B-E8) or of control
U
purified murine serum IgG (Sigma Chemicals, St Louis, MO) was
c
-m
added at initiation of the cultures. To reverse the effect of anti-ILA
6 MoAb, the MoAb was incubated with human rIL-6 (100 ng/mL)
V
g p n , E for
~ 2 ghours
~ before
~ being added to cultures. On day 5, the percentages
S
S
of
myeloma
cells
and myeloma cells in S phase were determined as
T)
described above.
8
W
Evaluation of circulating anti-IL-6 MoAb levels. Levels of cirU
culating anti-IL-6 MoAb were determined by enzyme-linked immum
nosorbent assay (ELISA). Polystyrene plates (Immuno I; Nunc,
.*
Kamstrup, Denmark) were coated overnight with purified goat IgG
d
B
against mouse Ig (10 pg/ml in phosphate-buffered saline [PBS];
Tag0 Inc, Burlingame, CA) at room temperature. Plates were satu+m
c . Em
rated with 1% bovine milk proteins (BMP) in PBS for I hour. After
>ai
E 5.5
five washes in PBS containing 0.05% Tween 20 (Sigma Chemicals),
I S $;
100 pL of the patient plasma diluted in PBS-BMP-Tween (1 % BMP
-m8: -e 8m
in PBS containing 0.05% Tween 20) was added for 1 hour at room
5 E
n S
temperature. Plates were washed five times with PBS-BMP-Tween
a> gc
before a solution containing peroxidase-conjugated goat antimouse
C
._
IgG (Jackson Laboratories) was added for 1 hour and then washed
*- 5
again five more times. Reactivity was determined by the intensity
c c
of the enzymatic reaction to substrate 0-phenylene diamine (for 20
;F
d
t d
minutes) measured by absorbance at 492 nm (Titertek Multiskan
MC; Flow, Irvine, UK) after the addition of 50 pL 2 N HzS04.
c u
a .
.Mouse Ig concentrations were calculated using a reference curve
t l B
-ya ?dr ga obtained with serial dilution of mab-8 or B-E8 MoAb.
U c =
orno
Detection of human antibodies to anti-IL-6 MoAb. The presb
ence of human antibodies to anti-IL-6 MoAb was also checked by
: . G
ELISA. Plates (Immuno I; Nunc) were coated with either 10 &nL
Lo
2.5;
(in PBS overnight at room temperature) of mab-8, B-E8, or a control
wrja
MoAb of the same isotype (CD37, BL14) as IgGI. To ensure that
- c:z
9 .g $ .f
the plates were coated with thesame amount of each MoAb a peroxi'C
52 .;gn
dase-conjugated goat antimouse IgG (Jackson Laboratories) was
used. Plates were saturated with PBS-BMP, and patient's serum
P O ai t
r
samples (diluted 1/50 in PBS-BMP) were added for l hour at room
Er0
g: G 3 8
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ANTI-IL-6 THERAPY IN MYELOMA
687
Table 2. R e s u b of Anti-11-6 Therapy in 7 Patients Receiving More Than 1 Week of Treatment
Responsive to Anti-ILG MoAb
Pretreatment Features
Patients treatedmore than
1 week
Ig type, light chain
subtype
Types of tumors
Presenting fever
(correction with MoAb)
Severe subsequent
infection
Hypercalcemia (correction
with MoAb)
Platelet counts (lO'/pL) (%
maximum suppression)
Neutrophil counts/pL (%
maximum suppression)
CRP serum levelst (rng/L)
4
5
Unresponsive to Anti-IL-6 MoAb
6
7
8
9
10
AK
Sec. PCL
AK
MM
AK
MM
GK
MM
GK
Primary PCL
GX
AK
Sec. PCL
Sec. PCL
-
-
+ (yes)
-
-
+ (yes)
+ (yes)
-
+
-
-
+ (death)
-
-
-
+ (no)
+ (partial control) + (no)
+ (no)
+ (no)
+ (no)
1 4 8 (45%)
272 (86%)
184 (56%)
1,845 (80%)
2,800 (78%)
3,000 (65%)
88 (52%)
4,300 (95%)'
57 (37%)
17 (40%)
122 (35%)
3,784 (stable) 2,500 (70%) 2,700 (stable)
(% maximum
suppression)
5 (100%)
21 (100%)
90 (100%)
46 (96%)
90 (75%)
60 (88%)
123 (60%)
Undetectable ( < l mg)
Daily in vivo IL-6
production (pg)
Overall changes of
myeloma Ig
Documented
antiproliferative effects
on myeloma cells
Not done
1.7
4
z 46
>l65
>47
>341
\
/
Yes
Survival (dl
-30%
/
93
Yes
45
Yes
128
/
/
No
No
17
11
/
/
No
17
No
13
Due to toxic chemotherapy.
t Pretreatment values-CRP has been monitored as shown in the Fig 2. Platelet and neutrophil counts were monitored every day until death
(patients no. 7, 8, 9, and 10). then every other day, then twice a week,
temperature. Human Ig were detected by incubating plates with perundetectable levels in 1 patient (no. 9). Human antibodies
oxidase-conjugated goat antihuman IgG or IgM (Jackson Laborato murine B-E8 MoAb were observed in 2 patients (no. 8
tories). Reactivity was determined as above.
and 9) on days 9 to 1 1 resulting in rapid clearance of circulatIL-6 bioassay and source of IL-6. Biologic activity of IL.-6 was
ing anti-IL-6 MoAb despite continued drug administration
as previously de~cribed.~
evaluated using the B9 hybridoma bioassay
in one patient (Fig 1). No anti-B-E8 or anti-mab-8 activity
B9 hybridoma was a generous gift of L. Aarden (Amsterdam, The
was found in the other patients' serum. For patient no. 6,
Netherlands).
the data have been detailed elsewhere.I6
Evaluation of IL-6productionin vivo. The overall daily production of IL-6 was evaluated according to a methodology previously
Effects on General Clinical and Biologic Symptoms
described by ~ u r s e l v e s . It
' ~is~based
~
on the observation that the
anti-IL-6MoAbinducesIL-6tocirculateinthe
form ofstable
The results are outlined in Table 2. There were no lifemonomeric immune complexes and protect it from rapid clearance
threatening side effects.
in vivo. In patients with partial inhibition of
CRP production, the
Clinical effects. Four patients (no. 1, 6, 9, and 10) with
consumption of L-6by cells was not completely inhibited and the
low-grade fever ranging from 37°C to 38"C, without evitrue in vivo production was compulsorily greater than our evaluation.dence of overt infection showed a normalization in body
The ability of neutralizing IL-6 by the anti-L-6 MoAb was estitemperature within 2 days of treatment with anti-L-6
mated fro the abaques we previously published.''
MoAb. This was frequently associated with disappearance
of fatigue and a slight but significant improvement of the
RESULTS
performance status and pain.
Anti-IL-6 MoAb Serum Concentrations and Immunization
BioEogic effects. Except patient no. 2 (shortest treatment)
In all patients receiving systemic therapy, serum anti-ILand 3 (local treatment), all the patients developed further
6 MoAb (approximately 1 pg/mL) was detectable 24 hours
worsening of thrombocytopenia (35% to 86%; median,
after the first IV injection and increased progressively to an
40%). One patient with marked thrombocytopenia (no. 9,
average value of 5 pg/mL on days 2 to 4 and to 5 to 17 pg/
17,OOO/pL) before therapy required platelet transfusion. Six
mL on days 7 and 8. No B-E8 MoAb was detected in the
of these 8 patients also showed neutrophil reduction (65%
serum of the patient receiving intrapleural injections during
to 95%), including 1 due to toxic antibiotherapy (patient
the first 2 days. Concentrations of 6 to 17 pg/mL were susno. 7). Three patients (no. 2, 5, and 8) developed bacterial
tained in 5 patients (no. 4,5 , 7, 8, and 10) and declined to
infections, which were lethal in patients no. 2 and 8. The
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688
BATAILLE ET AL
0B - E 8
Table 3. Inhibition of Myeloma Cell Growth (Labeling Index)
by the Anti-IL-6 MoAb In Vitro
INJECTIONS
~
anti-B-E8
ANTIBODIES
Patient
No.
10
1
2
-
h
E
3
P
v
m
W
A
6
7
8
5
Sample Origin
Blood
Bone marrow
Bone marrow
Peripheral blood
Pleural effusion
Bone marrow
Bone marrow
Peripheral blood
Controls
30
21
18
16
16
8
1
1.5
~~
With
Anti-IL6 MoAb
With Anti-IL-6
MoAb and IL6
0.7
0
0.6
1
0.5
17
21
8
12
8
10
1.5
1.5
0.3
0.5
0
Values are the percentage of myeloma cells in
tients, Materials, and Methods.
S phase. See Pa-
0
DAY
OF TREATMENT
Fig 1. Clearance of B-E8 MoAb after anti-B-E8 immunization in
patient no. 9.
neutropenia was not different in these patients from that of
the others.
Because C-reactive protein (CRP) production by freshly
explanted human hepatocytes is dependent on IL-6 in vitro,"
CRP serum levels were determined before and during intravenous anti-IL-6 MoAb. Before treatment, CRP serum levels were elevated in all the patients, ranging from 5 to 160
mg/L (median, 60 mg/L; normal values, < 1 mg/L by nephelometry). A significant reduction in CRP ofat least 50%
was observed in all the patients receiving anti-IL-6 therapy.
Sustained suppression to undetectable levels (< 1 mg) was
observed in 3 patients (no. 4, 5, and 6) receiving more than
1 week of therapy (Fig 2). In the other 4 cases (no. 7, 8, 9,
and lo), CRP serum levels were reduced by 60% to 96%
but remained above normal levels (Fig 2 and Table 2). It is
noteworthy that the maximum CRP serum level reduction
.-A
120
t Case5
n
\
E
v
v)
60
+-
t Case8
Case 7 -a-
Case 10
LL
oi
W
40
20
0
0
2
4
6
8
l
0
1
2
1
4
DAYS OF TREATMENT
Fig 2. Significant reduction of serum CRP levels in 6 patients with
a more than 1 week of treatment with theB-E8 anti-IL-6 MoAb.
correlates with the maximum and absolute platelet counts
reduction (Table 2).
The daily production of IL-6 has been estimated as previously described by ourselves.2oData are not evaluable in
patients no. 1 and 2 (too short a treatment) and not available
in patient no. 4. For the others, the daily IL-6 production
ranged from 1.7to more than341 pgld. Of noteisthat
CRP was totally inhibited in patients with the lowest IL-6
production (no. 5 and 6, Table 2).
Anti-proliferative Effects of Anti-IL-6 MoAb
In vitro studies. Mononuclear cells from bonemarrow
(no. 2, 3, 5 , and lo), blood (no. I , 3, 8, and 9), and pleural
effusion (no. 3) samples were cultured as previously described in Patients, Materials, and Methods with anti-IL-6
MoAb or anti-IL-6 MoAb and IL-6; mab-8wasusedin
patient no. 1 and B-E8 in the other cases. In all patients
exhibiting S phase values greater than 1% in vivo, complete
inhibition o myeloma cell proliferation was observed (Table
3) as previously published by ourselves inmany patients
with
Similar results were previously published for
patient no. 6.16In agreement with these results, IL-6-dependent cell lines were established in 4 patients (no. 1, 3, 7,
and 8).
In vivostudies.
Concomitantly withinvitro
investigations, in vivo cytokinetic studies were performed in patient
no. 3 while receiving anti-IL-6 MoAb intrapleurally for a
malignant pleural effusion and in patients no. 1, 4, 5 and 6.
Patient no. 3 with primary PCL exhibited primary treatment drug resistance with subsequent development of a malignant pleural effusion. Upon local administration of 20
mg of B-E8 anti-IL-6 MoAb on 3 successive days, a 80%
decrease of the concentration of tumor cells and of their
proliferative compartment (from 16% to 2.5% myeloma cells
in S phase) was observed (Fig 3A). No diffusion of the antiIL-6 MoAb was found outside the pleural fluid. In this case,
the local concentration of MoAb was 20 pglmL on day 1 of
treatment, increasing up to 60 pg1mL on day 3. This finding
was in agreement with a diffusion volume of 1 L. In these
conditions, this in vivo model was very close to an in vitro
situation. The dramatic decrease of myeloma cell proliferation and of myeloma cell counts during treatment was
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
689
ANTI-IL-6 THERAPY IN MYELOMA
cell proliferation decreased markedly in vivo, reaching a low
S-phase fraction of 3% after 4 days (Fig 3B). The absolute
increase in circulating myeloma cells to 55,OOO/pL throughout the 4 days of treatment with anti-IL-6 may have resulted
from sustained tumor cell proliferation, albeit at a markedly
reduced level of 3% versus 30% at the onset of therapy.
Patient no. 2 died quickly and was not evaluable for antiproliferative effects of anti-IL-6 MoAb. In patients no. 4,
5, and 6, a transient but significant reduction of myeloma
cell labeling index was obtained within the bone marrow. In
patient no. 4, a reduction from 19% to 3% of the myeloma
bone marrow labeling index was observed, with a subsequent
increase to 14%. In patients no. 5 and 6, a reduction from
1.5%and 4.5%, respectively, to 0% was noted. Disease progressed in the other 4 patients and no antiproliferative effect
was documented.
Antitumoral Effects of Anti-[L-6 MoAb
-7 -6 -5 -4 -3 -2 -1 0 l 2 3 4 5
DAYSOFTREATMENT
Fig 3. (A) Significant reduction in both myeloma cell counts and
myeloma celk in S phase in patient no. 3 during B-E8 anti-IL-6 therapy. (B)Significant reductionin thepercentages ofmyeloma cells in
S phase in patient no. 1 during mab-8 anti-ll-6 therapy.
therefore in agreement with the inhibition of myeloma cell
proliferation by the anti-IL-6 MoAb that was observed in
short-term cultures in vitro in this patient (Table 3).
Patient no. 1 developed secondary PCL, with 11,200 myeloma cells/mL in peripheral blood and an S phase fraction of
30%. In our experience and that of others, such proliferative
activity in peripheral blood was quite unusual. Considering
this high proliferative activity and that B-E8 MoAb was
administered intravenously, thus in the close contact of tumor cells, the situation was very similar to that of patient
no. 3 and that ofin vitro studies. Upon administration of
gradually increasing doses of mab-8, in vivo drug levels
increased progressively to 7.7 pg/mL on day 4 and myeloma
None of the patients treated had improved outcome or
achieved remission as defined by standard clinical response
criteria for MM. The observed effects after anti-IL-6 administration in this poor-risk patient population must therefore
be categorized as biologic effects. Indeed, although tumor
cell proliferation was inhibited in peripheral blood in 1 patient (no. l ) and in pleural effusion in another (no. 3), both
died within 1 week due to their terminal disease stage. A
third patient (no. 2) had central nervous system involvement
and died on day 5. The remaining patients (no. 4 through
10) were treated for 13 to 68 days (median, 17 days). As
emphasized inthe previous section, a clear-cut objective
antiproliferative effect was observed on malignant cells in
vivo in 3 patients (no. 4, 5, and 6). Among them, 1 patient
(no. 6) achieved 30% tumor cytoreduction for 2 months, but
relapse occurred after stopping anti-IL-6 MoAb. Although
patients no. 4 and 5 had significant (but transient) reduction
in their marrow plasma cell labeling index, disease progressed. Furthermore, fast disease progression was observed
in 4 other patients (no. 7, 8, 9, and 10). CRF' synthesis was
transiently inhibited in patient no. 9, in association with
reduction of overall white blood cells and plasmablasts (86%
inhibition) between days 3 and 6. However, a progression
occurred on day 7 in conjunction with the emergence of
human antibodies to Fc fragments of murine B-E8 MoAb
that was no longer detectable. Of major interest (and as
outlined in Table 2 ) a clear correlation was found between
in vivo daily IL-6 production, inhibition of CRP synthesis,
and antiproliferative effects. Such effects were onlyobserved
in patients no. 4 5 ,and 6, presenting the lowest IL-6 production. In these 3 patients, the B-E8 MoAb was able to inhibit
CRF'. In the 4 other patients presenting the highest production of IL-6, the B-E8 MoAb wasunableto
completely
inhibit CRP synthesis and thus IL-6. These results are summarized in Table 2.
DISCUSSION
In vitro studies performed in our patients receiving antiIL-6 MoAb in vivo simply and clearly show that, among
the various putative myeloma cell growth factors, IL-6 plays
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690
an essential role .in controlling spontaneous myeloma cell
proliferation in vitro?’ Indeed, adding anti-IL-6 MoAb dramatically inhibited the spontaneous proliferation of myeloma
cells that occurred in 5-day culture^.^.^ These in vitro concentration of anti-IL-6 MoAb was sufficient to neutralize 95%
of the IL-6 produced in the short-term cultures. Moreover,
IL-6-dependent myeloma cell lines have been obtained for
some of these patients.*
To discuss the effects of anti-IL-6 MoAb in vivo, one
has first to assess the feasibility to block IL-6 activity in the
close vicinity of tumor cells. The answer to this question
has been made easy by our previous findings that anti-IL6 MoAb induced large amounts of IL-6 to circulate in the
form of stable immune complexes that have the same halflife as the free antibody (3 to 4 days).” Thus, when stable
plasma concentrations of anti-IL-6 MoAb and of IL-6/antiIL-6 MoAb complexes are achieved (after 6 to 8 days of
treatment), one can predict the ability of the anti-IL-6 MoAb
to block IL-6 binding to the cell surface high-affinity IL6 receptors according to the methodology we previously
published.19”0Interestingly, these predictions fitted perfectly
well the inhibition of CRP production observed in these
patients. CRP is an acute-phase protein whose production
by human hepatocytes in primary cultures is controlled by
IL-6.” The present study shows that, in patients no. 5 and
6 producing low amounts of IL-6 (<4 pgld), a complete
inhibition of CRP production was found, unlike in patients
with larger IL-6 production (>46 pg/d up to >300 pgld).
Actually, mathematical modelling predicts an inability to
block IL-6 as soon as IL-6 production is greater than 18 pg/
d (B. Klein, unpublished observations). The good value of
CRP for predicting the effects of a n t i - L 6 therapy based
on the plasma concentrations of MoAb and IL-6/anti-IL-6
MoAb complexes is understandable. Indeed, IL-6 is mainly
produced in the tumor environment in our patients treated
with anti-IL-6 MoAb. It is carried to the liver in the form
of monomeric complexes so that the ratio of the concentrations of these complexes to those of free MoAb should be
similar to the ratio found in the plasma. In the tumoral samples, the situation should be different. As IL-6 is produced
in the tumoral samples and as the anti-IL-6 MoAb has to
diffuse to the tumoral sample, the ratio of IL-6/anti-IL-6
MoAb complexes to free MoAb is compulsorily superior to
that found in the plasma. In other words, it will be much
more difficult to neutralize IL-6 in the tumor sample than to
neutralize CRP production in the liver.
In this study, we got the opportunity to evaluate the antiproliferative effects of anti-IL-6 MoAb knowing the concentrations of IL-6 and anti-IL-6 MoAb close to proliferating tumor cells. In patient no. 3, the anti-IL-6 MoAb was
injected directly in the pleural effusion and did not diffuse
outside the tumor site. In patient no. 1, all circulating tumor
cells were proliferating (30% myeloma cells in the S phase).
In these 2 cases, a dramatic inhibition of tumor proliferation
was observed as it was observed for the same tumor cells
in vitro. In the other patients, even as CRP production was
not completely inhibited, it is easily understandable that no
major antitumor activity was found. Thus, our study shows
that, as soon as the anti-IL-6 MoAb is in sufficient amount
BATAILLEET
AL
to neutralize IL-6 activity in the close vicinity of the tumor
cells, a dramatic inhibition of myeloma-cell proliferation is
observed in vivo as it was observed in vitro. However, none
of the patients treated had improved outcome or achieved
remission as defined by standard clinical response criteria
for MM. The observed effects after anti-IL-6 administration
in this poor-risk patient population must therefore the categorized as biologic effects.
Among the other beneficial effects of anti-IL-6 MoAb
observed, resolution of fever and hypercalcemia (data not
shown) are of particular importance. The well-domumented
pyrogenic properties of IL-6’ were clearly shown in those
patients with low-grade fever whose body temperature
quickly normalized under anti-IL-6 therapy. Recent evidence shows that IL-6 also functions as a bone-resorbing
factor especially in association with its re~eptor.’~
A hypocalcemic effect was observed in a responsive patient, indicating
that IL-6 might play a role in MM-induced bone resorption.
Among the side effects observed, thrombocytopenia is of
interest. Initial thrombocytopenia was observed in most of
patients of this series. Subsequently to anti-IL-6 therapy, a
further decline in platelet counts was observed in all cases.
The involvement of IL-6 in primate thrombopoiesis is well
but the exact role of this cytokine in humans, in association with other cytokines, is not completely
defined.
It is noteworthy that only 1 of 7 patients evaluated for
human antibodies to murine anti-IL-6 MoAb showed strong
immunization responsible for complete clearance of B-E8
anti-IL-6 MoAb and resulting in fast progression. Weak
immunization was observed in patients no. 4,5, and 6 who
received the longest treatments.
In conclusion, our data show that administration of sufficient quantities of anti-IL-6 MoAb, resulting in inhibition
of CRP production and neutralization of IL-6 production in
vivo can block, at least transiently, the proliferation of the
myeloma plasmablastic compartment. They confirm thatIL6 is an essential myeloma cell growth factor in vivo. However, many of our patients had severe disease and produced
huge amounts of IL-6, making anti-IL-6 MoAb unable to
neutralize them. Thus, clinical trials in patients in earlier
disease stages are warranted. Considering the risk of immunization against the murine anti-IL-6 MoAb, humanized
anti-IL-6 MoAb might prove useful, especially for treatment
of patients with earlier stage disease.
ACKNOWLEDGMENT
We thank L. Bataille-Zagury for editing the English text and A.
Bauhain and P. Doiteau for typing the manuscript.
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1995 86: 685-691
Biologic effects of anti-interleukin-6 murine monoclonal antibody in
advanced multiple myeloma
R Bataille, B Barlogie, ZY Lu, JF Rossi, T Lavabre-Bertrand, T Beck, J Wijdenes, J Brochier and B
Klein
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