by guest
`
`
`
`www.bloodjournal.orgFrom
`
`on May 22, 2017.
`
`For personal use only.
`
`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 levels are associated with plasmablastic pro-
`liferative 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 re-
`ceived intrapleural therapy. Of the 3 patients who suc-
`cumbed t o progressive MM after less than 1 week of treat-
`ment (including the only 1 treated locally), 2 with evaluable
`data exhibited marked inhibition of plasmablastic proliiera-
`tion. Among the 7 patients remaining more homogeneous
`receiving the anti-IL-6 MoAb for more than 1 week, 3 had
`objective antiproliierative effect marked by a significant re-
`duction of the myeloma cell labelling index within the bone
`marrow. One of these 3 patients achieved a 30% regression
`of tumor mass. However, none of the patients studied
`
`achieved remission or improved outcome as judged by stan-
`dard clinical criteria. M major interest, objective antiprolifer-
`ative effects were associated with complete inhibition of
`C-reactive protein (CRP) synthesis and low daily 11-6 produc-
`tion 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 to 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 was associated
`with dramatic progression. These data show that anti-lL-6
`MoAb can suppress the proliieration of 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 granulo-
`poiesidthrombopoiesis.
`0 1995 by The American Society of Hematology.
`
`I
`
`standard chemotherapy and with a life expectancy less than 1 month
`NTERLEUKIN-6 (&6), a pleiotropic cytokine produced
`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
`mechanisms and acute-phase responses.’ pioneering studies
`in the current study, considering new data on L - 6 production in
`have shown that this cytokine controlled the proliferation2” but
`vivo.I6 Written informed consent was obtained from all the patients.
`did not induce the differentiation6v7 of immature myeloma cells
`Selected pretreatment characteristics of the 10 patients are summa-
`in vitro. First, anti-L-6 MoAb blocked the spontmwus prolif-
`rized in Tables 1 and 2. All but 1 patient were treated with intrave-
`eration 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 addition of exogeneous L - 6 further increased it.s5 Second,
`mg/day for at least 4 days (up to 68 days). One patient (no. 3)
`myeloma cell lines whose proliferation is dependent on addition
`received B-E8 intrapleurally for a malignant pleural effusion at a
`from every patient
`of exogenwus IL-6 have been obtained
`daily dose of 20 mg for 3 days. Response to treatment was evaluated
`with extramedullary proliferation? All studies agreed that large
`using standard criteria, including performance status, body tempera-
`ture, weight, and improvement of symptoms such as anemia, hyper-
`amounts of IL-6 are produced by the tumoral microenvironment
`calcemia, 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 production of IL-6 by the myeloma
`this production was only very
`cells themselves?.” However,
`minor (1/1,OOO) as compared with that of the tumoral microen-
`vironment. For these reasons, these studies could not exclude
`a minor contamination by environmental cells or more simply
`a weak activation of L-6 gene in myeloma cells by exogeneous
`IL-6, as has been previously reported.” The involvement of
`of in-
`this cytokine in vivo is supported by the relationship
`creased L - 6 serum levels to in vivo tumor cell kinetics, disease
`severity, and survival
`of patients with
`multiple myeloma
`These data led us to develop a clinical trial to define
`the effects of a murine anti-L-6 monoclonal antibody (MoAb)
`in patients with advanced MM (mainly plasma cell leukemia).
`The rationale of this project was also supported by data obtained
`in mice showing that (1) L-6 is a major paracrine plasmacy-
`tom growth factor and that (2) anti-L-6 and anti-L6 recep-
`tor MoAbs significantly inhibit the in vivo expansion of plas-
`macytomas and improve the survival of inoculated mice.I5
`PATIENTS, MATERIALS, AND METHODS
`
`Materials and Methods
`Injection of anti-IL-6 MoAb was
`Infection ofanti-lL-6 MoAb.
`performed in vivo as previously described.16 Nine of the 10 patients
`
`Laboratoire Central d’Hdmatologie et Laboratoire
`From the
`d’Oncoginkse Immunohimatologique-lnstitut de Biologie, Nantes,
`France; Arkansas Cancer Research Center, Little Rock, AR; IN-
`SERM U.291, lmmunopathologie des Maladies Tumorales et Auto-
`immunes, Montpellier, France; and Innothkrapie, Besancon, France.
`Submitted June 16, 1994; accepted February 27, 1995.
`Supported by grants from I’Association Pour la Recherche sur le
`Cancer, la Ligue Nationale de Lutte contre le Cancer (Paris,
`France), and la Ligue Rigionale de Lutte contre le Cancer (Nantes,
`France).
`Address reprint requests to Rigis Bataille, MD, PhD, Laboratoire
`central d’Hkmatologie, Institut de Biologie, 9 quai Moncousu, 44035
`Nantes Ckdex 01, France.
`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
`
`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
`
`685
`
`IPR2018-00685
`Celgene Ex. 2036, Page 1
`
`

`

`686
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`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`on May 22, 2017.
`
`For personal use only.
`
`BATAILLE ET AL
`
`2
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`received B-E8 and 1 received mab-8 MoAb, which were adminis-
`tered according to a schedule outlined in Table 1. Doses of anti-
`IL-6 MoAb were selected to provide serum concentrations similar
`to those known to inhibit myeloma cell proliferaion in vitro and
`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 Ser-
`vice, Amsterdam, The Netherlands), respectively, by immunizing
`mice with rIL-617,1* These MoAb were purified and
`submitted to
`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. Periph-
`eral blood or bone marrow samples for all studies, including pharma-
`cokinetic studies, were collected at 7 AM, 1 hour before mab-8 or
`B-E8 injections. Peripheral blood serum and plasma were stored at
`-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 percent-
`ages of myeloma cells were determined by cytoplasmic immunoflu-
`-
`orescence using anti-K or anti-A light chain antibodies directly cou-
`pled to
`fluorescein (Kallestadt, Austin, TX). The percentages of
`C .- e
`plasma cells in S phase (ie, the labeling index) were determined
`._
`using an antibromodeoxyuridine MoAb (Immunotech, Marseilles,
`U
`France) and a rhodamine-labeled goat antimouse Ig (Jackson Labora-
`-
`0
`D
`tories, West Grove, PA) in a double fluorescence technique described
`c
`m
`elsewhere.' Cells were cultured at lo6 cellslml in RPM1 1640 me-
`5
`L 5 2 2
`dium supplemented with 5 X lO-5 m o m 2ME and 5% fetal calf
`s d
`s L m
`serum for 5 days, hence referred to as culture medium. In some
`S
`groups, 10 pg/mL of anti-IL-6 MoAb (mab-8 or B-E8) or of control
`'c
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`added at initiation of the cultures. To reverse the effect of anti-IL-
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`A
`g $ . ~ ~ g ~ ~ e ~ g p , E ~ g ~ ~
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`m E m t n
`v = m D t u n a r n n
`for 2 hours before being added to cultures. On day 5, the percentages
`S
`9 9
`S
`u u
`of myeloma cells and myeloma cells in S phase were determined as
`described above.
`8
`Evaluation of circulating anti-IL-6 MoAb levels. Levels of cir-
`culating anti-IL-6 MoAb were determined by enzyme-linked immu-
`nosorbent assay (ELISA). Polystyrene plates (Immuno I; Nunc,
`Kamstrup, Denmark) were coated overnight with purified goat IgG
`against mouse Ig (10 pg/ml in phosphate-buffered saline [PBS];
`Tag0 Inc, Burlingame, CA) at room temperature. Plates were satu-
`rated with 1% bovine milk proteins (BMP) in PBS for I hour. After
`five washes in PBS containing 0.05% Tween 20 (Sigma Chemicals),
`100 pL of the patient plasma diluted in PBS-BMP-Tween (1 % BMP
`in PBS containing 0.05% Tween 20) was added for 1 hour at room
`temperature. Plates were washed five times with PBS-BMP-Tween
`before a solution containing peroxidase-conjugated goat antimouse
`IgG (Jackson Laboratories) was added for 1 hour and then washed
`again five more times. Reactivity was determined by the intensity
`of the enzymatic reaction to substrate 0-phenylene diamine (for 20
`minutes) measured by absorbance at 492 nm (Titertek Multiskan
`MC; Flow, Irvine, UK) after the addition of 50 pL 2 N HzS04.
`Mouse Ig concentrations were calculated using a reference curve
`obtained with serial dilution of mab-8 or B-E8 MoAb.
`Detection of human antibodies to anti-IL-6 MoAb. The pres-
`ence of human antibodies to anti-IL-6 MoAb was also checked by
`ELISA. Plates (Immuno I; Nunc) were coated with either 10 &nL
`(in PBS overnight at room temperature) of mab-8, B-E8, or a control
`MoAb of the same isotype (CD37, BL14) as IgGI. To ensure that
`the plates were coated with the same amount of each MoAb a peroxi-
`dase-conjugated goat antimouse IgG (Jackson Laboratories) was
`used. Plates were saturated with PBS-BMP, and patient's serum
`samples (diluted 1/50 in PBS-BMP) were added for l hour at room
`
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`IPR2018-00685
`Celgene Ex. 2036, Page 2
`
`

`

`by guest
`
`
`
`www.bloodjournal.orgFrom
`
`on May 22, 2017.
`
`For personal use only.
`
`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
`
`Pretreatment Features
`Patients treated more 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)
`(% maximum
`suppression)
`
`Responsive to Anti-ILG MoAb
`
`Unresponsive to Anti-IL-6 MoAb
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`GK
`MM
`
`-
`+
`
`+ (no)
`
`GK
`Primary PCL
`
`+ (yes)
`
`GX
`Sec. PCL
`
`AK
`Sec. PCL
`
`-
`
`-
`
`AK
`Sec. PCL
`+ (yes)
`
`-
`
`-
`+ (partial control) + (no)
`
`+ (death)
`
`-
`
`+ (no)
`
`+ (no)
`
`+ (no)
`
`AK
`MM
`
`+ (yes)
`
`-
`
`4
`
`AK
`MM
`
`-
`
`-
`
`-
`
`1 4 8 (45%)
`
`272 (86%)
`
`184 (56%)
`
`88 (52%)
`
`57 (37%)
`
`17 (40%)
`
`122 (35%)
`
`1,845 (80%)
`
`2,800 (78%)
`
`3,000 (65%)
`
`4,300 (95%)' 3,784 (stable) 2,500 (70%) 2,700 (stable)
`
`5 (100%)
`90 (100%)
`21 (100%)
`Undetectable ( < l mg)
`
`46 (96%)
`
`90 (75%)
`
`60 (88%)
`
`123 (60%)
`
`Daily in vivo IL-6
`production (pg)
`Overall changes of
`myeloma Ig
`Documented
`antiproliferative effects
`on myeloma cells
`Survival (dl
`Due to toxic chemotherapy.
`as shown in the Fig 2. Platelet and neutrophil counts were monitored every day until death
`t Pretreatment values-CRP has been monitored
`(patients no. 7, 8, 9, and 10). then every other day, then twice a week,
`
`Not done
`
`1.7
`
`/
`
`/
`
`Yes
`93
`
`Yes
`45
`
`4
`
`\
`-30%
`
`Yes
`128
`
`z 46
`
`/
`
`No
`
`17
`
`>l65
`
`/
`
`No
`1 1
`
`> 47
`
`/
`
`No
`17
`
`>341
`
`/
`
`No
`13
`
`undetectable levels in 1 patient (no. 9). Human antibodies
`to murine B-E8 MoAb were observed in 2 patients (no. 8
`and 9) on days 9 to 11 resulting in rapid clearance of circulat-
`ing anti-IL-6 MoAb despite continued drug administration
`in one patient (Fig 1). No anti-B-E8 or anti-mab-8 activity
`was found in the other patients' serum. For patient no. 6,
`the data have been detailed elsewhere.I6
`
`temperature. Human Ig were detected by incubating plates with per-
`oxidase-conjugated goat antihuman IgG or IgM (Jackson Labora-
`tories). Reactivity was determined as above.
`IL-6 bioassay and source of IL-6. Biologic activity of IL.-6 was
`evaluated using the B9 hybridoma bioassay as previously de~cribed.~
`B9 hybridoma was a generous gift of L. Aarden (Amsterdam, The
`Netherlands).
`Evaluation of IL-6production in vivo. The overall daily produc-
`tion of IL-6 was evaluated according to a methodology previously
`Effects on General Clinical and Biologic Symptoms
`
`described by ~urselves.'~~~ It is based on the observation that the
`form of stable
`anti-IL-6 MoAb induces IL-6 to circulate in the
`The results are outlined in Table 2. There were no life-
`monomeric immune complexes and protect it from rapid clearance
`threatening side effects.
`CRP production, the
`in vivo. In patients with partial inhibition of
`Clinical effects. Four patients (no. 1, 6, 9, and 10) with
`consumption of L-6 by cells was not completely inhibited and the
`low-grade fever ranging from 37°C to 38"C, without evi-
`true 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 esti-
`temperature 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
`performance status and pain.
`BioEogic effects. Except patient no. 2 (shortest treatment)
`and 3 (local treatment), all the patients developed further
`worsening of thrombocytopenia (35% to 86%; median,
`40%). One patient with marked thrombocytopenia (no. 9,
`17,OOO/pL) before therapy required platelet transfusion. Six
`of these 8 patients also showed neutrophil reduction (65%
`to 95%), including 1 due to toxic antibiotherapy (patient
`no. 7). Three patients (no. 2, 5, and 8) developed bacterial
`infections, which were lethal in patients no. 2 and 8. The
`
`RESULTS
`Anti-IL-6 MoAb Serum Concentrations and Immunization
`In all patients receiving systemic therapy, serum anti-IL-
`6 MoAb (approximately 1 pg/mL) was detectable 24 hours
`after the first IV injection and increased progressively to an
`average value of 5 pg/mL on days 2 to 4 and to 5 to 17 pg/
`mL on days 7 and 8. No B-E8 MoAb was detected in the
`serum of the patient receiving intrapleural injections during
`the first 2 days. Concentrations of 6 to 17 pg/mL were sus-
`tained in 5 patients (no. 4, 5, 7, 8, and 10) and declined to
`
`IPR2018-00685
`Celgene Ex. 2036, Page 3
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`
`on May 22, 2017.
`
`For personal use only.
`
`688
`
`BATAILLE ET AL
`
`0 B-E8 INJECTIONS
`a n t i - B - E 8 ANTIBODIES
`
`10
`h -
`
`E P
`
`v
`m
`W
`A
`
`5
`
`
`
`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 intra-
`venous anti-IL-6 MoAb. Before treatment, CRP serum lev-
`els were elevated in all the patients, ranging from 5 to 160
`mg/L (median, 60 mg/L; normal values, < 1 mg/L by nephe-
`least 50%
`lometry). A significant reduction in CRP of at
`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
`
`\
`
`t Case5 t Case8
`
`+- Case 7 -a- Case 10
`
`.-A n
`
`120
`
`E
`
`v
`v)
`
`LL oi
`W
`
`60
`
`40
`
`20
`
`0
`0
`
`2
`
`4
`
`l 0 1 2 1 4
`6
`8
`DAYS OF TREATMENT
`
`Fig 2. Significant reduction of serum CRP levels in 6 patients with
`a more than 1 week of treatment with the B-E8 anti-IL-6 MoAb.
`
`Table 3. Inhibition of Myeloma Cell Growth (Labeling Index)
`by the Anti-IL-6 MoAb In Vitro
`
`Patient
`No.
`
`1
`2
`3
`
`6
`7
`8
`
`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-IL-
`6 MoAb
`
`With Anti-IL-6
`MoAb and IL-
`6
`
`0.7
`0
`0.6
`1
`0.5
`0.3
`0.5
`0
`
`17
`21
`8
`12
`8
`10
`1.5
`1.5
`
`Values are the percentage of myeloma cells in S phase. See Pa-
`tients, Materials, and Methods.
`
`correlates with the maximum and absolute platelet counts
`reduction (Table 2).
`The daily production of IL-6 has been estimated as pre-
`viously described by ourselves.2o Data 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 1.7 to more than 341 pgld. Of note is that
`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 bone marrow
`(no. 2, 3, 5, and lo), blood (no. I, 3, 8, and 9), and pleural
`effusion (no. 3) samples were cultured as previously de-
`scribed in Patients, Materials, and Methods with anti-IL-6
`MoAb or anti-IL-6 MoAb and IL-6; mab-8 was used in
`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 in many patients
`with
`Similar results were previously published for
`patient no. 6.16 In agreement with these results, IL-6-depen-
`dent cell lines were established in 4 patients (no. 1, 3, 7,
`and 8).
`In vivo studies.
`investiga-
`Concomitantly with in vitro
`tions, 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 treat-
`ment drug resistance with subsequent development of a ma-
`lignant 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 anti-
`IL-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 prolif-
`eration and of myeloma cell counts during treatment was
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`ANTI-IL-6 THERAPY IN MYELOMA
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`689
`
`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 through-
`out 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 anti-
`proliferative 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 pro-
`gressed in the other 4 patients and no antiproliferative effect
`was documented.
`
`Antitumoral Effects of Anti-[L-6 MoAb
`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 admin-
`istration 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 pa-
`tient (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 in the 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 pro-
`gressed. 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 only observed
`in patients no. 4 5 , and 6, presenting the lowest IL-6 produc-
`tion. In these 3 patients, the B-E8 MoAb was able to inhibit
`CRF'. In the 4 other patients presenting the highest produc-
`tion of IL-6, the B-E8 MoAb was unable to
`completely
`inhibit CRP synthesis and thus IL-6. These results are sum-
`marized in Table 2.
`
`DISCUSSION
`In vitro studies performed in our patients receiving anti-
`IL-6 MoAb in vivo simply and clearly show that, among
`the various putative myeloma cell growth factors, IL-6 plays
`
`-7 -6 -5 -4 -3 -2 -1 0 l 2 3 4 5
`DAYSOFTREATMENT
`
`(A) Significant reduction in both myeloma cell counts and
`Fig 3.
`myeloma celk in S phase in patient no. 3 during B-E8 anti-IL-6 ther-
`apy. (B) Significant reduction in the percentages of myeloma 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 my-
`eloma 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 tu-
`mor cells, the situation was very similar to that of patient
`no. 3 and that of in 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
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`690
`
`an essential role .in controlling spontaneous myeloma cell
`proliferation in vitro?’ Indeed, adding anti-IL-6 MoAb dra-
`matically inhibited the spontaneous proliferation of myeloma
`cells that occurred in 5-day culture^.^.^ These in vitro concen-
`tration 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-IL-
`6 MoAb induced large amounts of IL-6 to circulate in the
`form of stable immune complexes that have the same half-
`life as the free antibody (3 to 4 days).” Thus, when stable
`plasma concentrations of anti-IL-6 MoAb and of IL-6/anti-
`IL-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 IL-
`6 receptors according to the methodology we previously
`published.19”0 Interestingly, 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 anti-L6 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 concentra-
`tions of these complexes to those of free MoAb should be
`similar to the ratio found in the plasma. In the tumoral sam-
`ples, 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 anti-
`proliferative effects of anti-IL-6 MoAb knowing the con-
`centrations of IL-6 and anti-IL-6 MoAb close to proliferat-
`ing 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
`
`BATAILLE ET 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 catego-
`rized 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 evi-
`dence shows that IL-6 also functions as a bone-resorbing
`factor especially in association with its re~eptor.’~ A hypocal-
`cemic 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 hu-
`mans, 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 suffi-
`cient 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 that IL-
`6 is an essential myeloma cell growth factor in vivo. How-
`ever, many of our patients had severe disease and produced
`huge a