Jean-Philippe Gaillard - Academia.edu (original) (raw)

Papers by Jean-Philippe Gaillard

Research paper thumbnail of Etude structurale et fonctionnelle du récepteur de l'interleukine-6 : interactions moléculaires dans la médiation du message IL-6

L'interleukine-6 (il-6) est une cytokine multifonctionnelle qui est impliquee comme facteur d... more L'interleukine-6 (il-6) est une cytokine multifonctionnelle qui est impliquee comme facteur de croissance dans les proliferations plasmocytaires benignes et malignes. Sa neutralisation a l'aide d'anticorps monoclonaux anti-il-6, entraine le bloquage du clone tumoral chez des patients atteints de myelome multiple (mm). Dans le but d'explorer la possibilite de bloquer le message il-6 en ciblant son recepteur, nous avons produit sept anticorps monoclonaux specifiques de la chaine gp80 du recepteur de l'interleukine-6 (il-6r). Nous avons recherche la fonction des epitopes cibles par nos anticorps. Les etudes d'inhibition des interactions ligand/recepteur et l'inhibition de la proliferation de lignees dependantes de l'il-6 nous ont montre que deux epitopes sont impliques dans la fixation de la cytokine et qu'un troisieme epitope est implique dans l'interaction des deux chaines du recepteur. L'association d'anticorps de specificite different...

Research paper thumbnail of Tumoral Sample Peripheral blood Pleural effusion Peripheral blood Pleural effusion Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Bone marrow and the percentages of myeloma cells

We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to b... more We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to be obtained from each of 10 tumor samples harvested from 9 patients with extramedullary proliferation. Fresh samples were cultured with interleukin 6 (IL-6) and granulocyte macrophagetolony stimulating factor (GM-CSF) at a high cell density and resulting HMCL growth became progressively dependent on IL-6 alone, no longer requiring GM-CSF. These HMCL, which had the same immunoglobulin gene rearrangements as the patients’ original myeloma cells, were designated XG-l to XG-9. XG HMCL had a plasma cell morphology, expressed plasma cell antigen (Ag), namely cytoplasmic immunoglobulins. CD38, 8-84 Ag, and CD77, and lacked the usual B-cell

Research paper thumbnail of IL-6-induced changes in synthesis of α 1-acid glycoprotein in human hepatoma Hep3B cells are distinctively regulated by monoclonal antibodies directed against different epitopes of IL-6 receptor (gp80)

European cytokine network

ABSTRACT

Research paper thumbnail of Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6

European cytokine network

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specif... more Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.

Research paper thumbnail of Gamma-interferon induces apoptosis of the B lymphoma WEHI-279 cell line through a CD95/CD95L-independent mechanism

European cytokine network

Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and ... more Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and natural killer (NK) cells, plays a central role in the development of humoral and cell-mediated immunity. IFN-gamma participates in the maturation and differentiation of B cells, but it has been previously reported that IFN-gamma may inhibit the early stages of B cell activation. We report that the inhibition of the B lymphoma cell WEHI-279-proliferation induced by IFN-gamma, involves the induction of typical features of apoptosis (nuclear chromatin condensation and fragmentation, cell shrinkage, phosphatidyl-serine (PS) exposure and mitochondrial membrane potential (delta psim) loss). IFN-gamma-mediated B cell apoptosis was decreased by the addition of the T helper type 2 cytokine, IL-4. WEHI-279 cells express CD95 and undergo apoptosis after treatment with either an agonistic anti-CD95 Ab or with a soluble recombinant CD95L. However, incubation with CD95-Fc or TRAIL-R1-Fc fusion protei...

Research paper thumbnail of Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma

Blood, Jan 15, 1994

We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to b... more We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to be obtained from each of 10 tumor samples harvested from 9 patients with extramedullary proliferation. Fresh samples were cultured with interleukin 6 (IL-6) and granulocyte macrophage-colony stimulating factor (GM-CSF) at a high cell density and resulting HMCL growth became progressively dependent on IL-6 alone, no longer requiring GM-CSF. These HMCL, which had the same immunoglobulin gene rearrangements as the patients' original myeloma cells, were designated XG-1 to XG-9. XG HMCL had a plasma cell morphology, expressed plasma cell antigen (Ag), namely cytoplasmic immunoglobulins, CD38, B-B4 Ag, and CD77, and lacked the usual B-cell Ag. They also expressed activation antigens such as CD28 with coexpression of CD28 and its ligand, B7 Ag, in four HMCL. Six HMCL expressed CD40, 4 CD23, and 5 its ligand, CD21. The XG HMCL bore adhesion molecules VLA-4 and CD44 (all 10 HMCL), VLA-5 (7 HMC...

Research paper thumbnail of Immunomodulating IL-6 activity by murine monoclonal antibodies

International journal of immunopharmacology, 1995

The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injec... more The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injection of murine monoclonal antibodies (MAb) directed against cellular targets, has been reported extensively in several studies. We analysed here HAMA in 12 patients (six with multiple myeloma, MM, and six with metastatic renal cell carcinoma, MRCC) who were treated with B-E8, an IgG1 MAb against interleukin-6 (IL-6). Efficiency of the treatment was evidenced by the drop in the serum levels of C reactive protein (CRP), of which the in vivo production is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All of immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MAb cleared rapidly from ...

Research paper thumbnail of Inhibiting IL-6 in human multiple myeloma

Current topics in microbiology and immunology, 1992

It has been well established by several groups that IL-6 is overproduced in patients with Multipl... more It has been well established by several groups that IL-6 is overproduced in patients with Multiple Myeloma (MM) and that this cytokine is an essential growth factor of myeloma cells both in vitro and in vivo (1–5). Most investigators also agree now that IL-6 is produced mainly by bone-marrow stromal cells (1, 6). The aim of this report was to summarize our recent results concerning the mechanisms of IL-6 production control by the bone-marrow environment and the different means of neutralizing IL-6 activity in vitro and in vivo.

Research paper thumbnail of Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum

European cytokine network, 1999

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specif... more Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3)...

Research paper thumbnail of Immunomodulating IL6 activity by murine monoclonal antibodies

International Journal of Immunopharmacology, 1995

The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injec... more The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injection of murine monoclonal antibodies (MAb) directed against cellular targets, has been reported extensively in several studies. We analysed here HAMA in 12 patients (six with multiple myeloma, MM, and six with metastatic renal cell carcinoma, MRCC) who were treated with B-E8, an IgG1 MAb against interleukin-6 (IL-6). Efficiency

Research paper thumbnail of Functional interaction of the gp80 and gp130IL-6 receptors in human B cell malignancies

Clinical and Laboratory Haematology, 1998

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggest... more IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin's malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recognizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the disappearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.

Research paper thumbnail of Dimerization and activation of the common transducing chain (gp130) of the cytokines of the IL-6 family by mAb

International Immunology, 1998

The objective of our research was to study the mechanisms of activation of mAb against the gp130 ... more The objective of our research was to study the mechanisms of activation of mAb against the gp130 transducer chain common to the IL-6 cytokine family. It has been found that among the 56 anti-gp130 available worldwide, none was able to activate the growth of IL-6-dependent myeloma cell lines. When certain of them were associated in pairs they allowed the cells to grow; alone, they were inhibitory. The same activation was also obtained by cross-linking certain anti-gp130 mAb on the cell membrane with a goat anti-mouse Ig antiserum. A bispecific mAb was prepared by the somatic fusion of two hybridomas secreting two mAb whose association was able to activate gp130 signaling; the bispecific mAb was inactive. The activating mAb were able to support long-term proliferation of the IL-6-dependent myeloma cell lines, which indicates that they are potential valuable growth factors of tumor cells and hematopoietic stem cells. When they were injected into SCID mice, they allowed human IL-6dependent myeloma cell lines to grow, develop tumors and metastasize. By studying the functional epitopes of the cell membrane gp130 receptors, it was shown that the activating mAb induced gp130 dimerization and STAT3 activation, as did IL-6.

Research paper thumbnail of Identification of a novel antigenic structure of the human receptor for interleukin-6 involved in the interaction with the glycoprotein 130 chain

Immunology, 1996

The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80)... more The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80) transmembrane chain (IL-6R) which associates with a signal-transducer gp130 chain. We previously raised a series of monoclonal antibodies (mAb) recognizing different epitopes of the human IL-6R and interfering with the function of the receptor. One of them, M182, was able to diminish the proliferation of IL-6-dependent plasmacytoma cell lines although it was found unable to inhibit the binding of IL-6 to its receptor. Using an enzyme-linked immunosorbent assay for measuring the binding of IL-6-IL-6R to the gpl30 chain, we showed that M182 was directed against a structure directly involved in the IL-6R/gpl 30 interaction. M 182 was able to potentiate the inhibitor effect of anti-IL-6R mAB which interfere with the binding of IL-6, leading to complete inhibition of the proliferation of IL-6-dependent cell lines. M182 was also found to synergize with inhibitory anti-IL-6 mAb. Therefore this structure appears to be an important regulatory domain of the IL-6R and a valuable target for inhibiting IL-6 signalling. INTRODUCTION Cell surface interleukin-6 (IL-6) receptors (IL-6R) belong to a class characterized by a complex hetero-oligomeric structure in which ligand-binding and signal-transduction are compartmentalized into distinct receptor units. Two transmembrane proteins are involved, a glycoprotein (gp)80 ligand-binding receptor (IL-6R) and an associated transducer gpl3O molecule.' Upon binding of IL-6 to its IL-6R, the IL-6-IL-6R complexes associate with the gpl30 molecules, resulting in the formation of high avidity IL-6-binding receptors and activation of transduction signals.' Membrane IL-6R is cleaved from the cell membrane2 or translated from an alternative spliced mRNA3'4 to give a gp55 soluble form (sIL-6R). It was demonstrated that both genetically engineering and plasma sIL-6R retained their capability to bind IL-6 and that the complexes formed were able to activate the gpl30 transducer chain.56 Therefore, in contrast with other soluble cytokine receptors which are generally antagonists,7 sIL-6R are able to promote IL-6 activity. IL-6 has recently been associated with a cell growth function of tumoral plasma cells in multiple myeloma.8'9

Research paper thumbnail of Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy

European Journal of Immunology, 1993

Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patien... more Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy" Soluble human interleukin-6 receptor (sIL-6R) was measured in the serum of 30 healthy individuals, 32 individuals with monoclonal gammopathy of undetermined significance (MGUS), 20 patients with early multiple myeloma (MM) and 54 patients with overt MM. The serum activity recognized by an immunoradiometric assay was determined to be s I L-~R , because of its binding capacity to IL-6 and its molecular mass of 55 kDa. All sera of healthy individuals contained sIL-6R (mean value: 89 ng/ml, range 17-300 ng/ml). Serum sIL-6R levels were increased by 51 % in patients with MGUS (mean value: 135 ng/ml, p C 0.005), by 44% in patients with early myeloma (mean value: 128 ng/ml, p C 0.001) and by 116 YO in patients with overt MM (mean value: 193 ng/ml,p < 0.001). In patients withMM, acomplete lackof correlation (p > 0.7) was found between serum sIL-6R levels and other previously recognized prognostic factors in this disease, particularly serum IL-6 levels and those factors related to tumor cell mass. The independence of serum sIL-6R levels on tumor cell mass was directly demonstrated by studying four patients with MM treated with autologous bone marrow transplantation for periods of between 320 and 760 days. These levels were found to be remarkably stable and constant, independent of whether patients relapsed or achieved complete remission. Finally, physiological concentrations of sIL-6R were found to increase by tenfold the sensitivity of human myeloma cell lines to L-6.These observations suggest a high control of the sIL-6R level in vivo, and, possibly, an important functional role of this circulating protein in patients with monoclonal gammopathies.

Research paper thumbnail of Identification of centerin: a novel human germinal center B cell-restricted serpin

European Journal of Immunology, 2000

Research paper thumbnail of Optimizing therapeutic strategies to inhibit circulating soluble target molecules with monoclonal antibodies: example of the soluble IL-6 receptors

European Journal of Immunology, 2001

Therapeutic targeting of soluble molecules such as cytokines can be achieved with monoclonal anti... more Therapeutic targeting of soluble molecules such as cytokines can be achieved with monoclonal antibodies (mAb). Anti-IL-6 mAb have been shown to form circulating complexes, resulting in the increase of the half-life of the cytokine in vivo. In IL-6-related diseases, the soluble human IL-6 receptors (shIL-6R), which have been shown to possess strong agonist activity, circulate in the plasma at a high concentration and must be neutralized. Their clearance was studied in mice that had been made to express circulating shIL-6R after i.p. grafting of mouse thymoma cells transfected with a gene coding for shIL-6R, treated with various anti-shIL-6R mAb recognizing different epitopes of the molecule. Injection of one anti-hIL-6R mAb stabilized the short-lived hIL-6R and led to their accumulation. The same result was observed when two mAb directed against two different epitopes of the hIL-6R were used. Clearance of the receptors was only achieved when three mAb specific for three different epitopes were injected. A permanent clearing of the hIL-6R could be obtained by repeated injections of the clearing mixture. No correlation was found between the ability of the mAb to clear the sIL-6R and to immunoprecipitate them in agarose gel. The F(ab') 2 fragments lost the clearing ability of the intact mAb. These results clearly show that therapeutic clearance of sIL-6R by mAb need at least three mAb directed against three different epitopes of the molecule, a conclusion which is likely to apply for clearing any soluble target molecule.

Research paper thumbnail of Activation of the gp130 signaling pathway by monoclonal antibodies directed against the gp130 molecule

European Journal of Immunology, 1997

Six cytokines of the interleukin (IL)-6 family involved in various inflammatory or tumoral diseas... more Six cytokines of the interleukin (IL)-6 family involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. We made a panel of anti-gp130 monoclonal antibodies (mAb) to study the structure and function of the gp130 molecule. These mAb recognized different epitopes of the gp130 that we called A to J. Most of the mAb were found to be inhibitors and we studied whether some of them could also induce gp130 activation. When used alone, none of them was able to initiate the proliferation of IL-6-dependent cell lines. However, some particular associations of the mAb were able to induce a proliferative response. mAb B1 could activate the lines in association with F1 or with I2 but not with I1, which in ELISA was similar to I2. In contrast mAb B2, which in ELISA appeared to be very similar to B1, was able to activate the cells in association with I1 but not with F1 or I2. Two other mAb belonging to specificities A and C were found to be activators either in association with I1 only, or with I1 or B2, respectively. These associations of mAb appeared to be nearly as potent activators as IL-6 itself. Although we still have no precise idea of the mechanisms involved, they are interesting tools to study the molecular interactions leading to gp130 activation and, from a practical point of view, valuable growth factors of hematopoietic stem cells.

Research paper thumbnail of Major role of the soluble interleukin-6/interleukin-6 receptor complex for the proliferation of interleukin-6-dependent human myeloma cell lines

European Journal of Immunology, 1997

Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activi... more Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activity for certain tumor cells such as plasma cells in multiple myeloma (MM). Upon binding of IL-6, soluble IL-6 receptor (sIL-6R) has been shown to retain its affinity for IL-6 and to associate with the signal-transducing gp130 chain. Therefore, contrary to the majority of soluble cytokine receptors, it plays an agonist role in IL-6 signaling. In order to test its physiological importance as compared to that of its membrane counterpart, we studied cells from two myeloma cell lines which need exogenous IL-6 to proliferate and release sIL-6R into their culture supernatant. Using a new culture system where the supernatant recirculated permanently through an anti-IL-6R affinity column, all sIL-6R was removed from the culture medium throughout the culture period. Under these conditions IL-6-dependent cells were unable to grow in the presence of physiological concentrations of IL-6, showing the major role of the sIL-6R for sustaining the proliferation of these cell lines. Increasing IL-6 concentrations well over the physiological values allowed the cells to proliferate again. No effect was seen when sIL-6R was removed from the supernatant of an IL-6-independent myeloma cell line. These results show that the levels of circulating sIL-6R (and thus those of IL-6/sIL-6R complex) are worth looking at in pathologies involving IL-6 hyperactivity.

Research paper thumbnail of SPECIFIC INHIBITION OF IL-6 SIGNALLING WITH MONOCLONAL ANTIBODIES AGAINST THE gp130 RECEPTOR

Cytokine, 1997

A family of cytokines [IL-6, IL-11, oncostatin M (OM), leukaemia inhibitory factor (LIF), ciliary... more A family of cytokines [IL-6, IL-11, oncostatin M (OM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1] involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. The complex formed with their specific receptors associates with a common transducing gp130 membrane protein (gp130) resulting in the formation of high avidity receptor and activation of tyrosine kinases. With the view of identifying gp130 domains specifically involved in IL-6 signalling, the authors prepared 37 new anti-gp130 mAb and analysed the structure-function relationship of the molecule. By cross-competition ELISA, the mAb were classified in 10 subgroups called A to J. By ELISA and BIAcore analysis, the mAb were found to recognize at least 18 antigenic specificities of the gp130 chain. The mAb reacted against the soluble and the membrane forms of gp130 as well. Their ability to inhibit the proliferation of the human myeloma cell line XG-4 of which the growth is strictly dependent on the presence of either exogenous IL-6, or LIF, or OM, or CNTF was studied. Besides mAb with no evident neutralizing effect (G and H) and mAb which neutralized equally well the activity of all tested cytokines (all mAb of groups A, I and J), some showed a selective effect. Those of group F inhibited also the proliferation induced by the 4 cytokines, but more specifically that dependent on the CNTF. mAb of groups B and E specifically inhibited the growth induced by IL-6, whereas those of group C inhibited that induced by LIF and OM. These results show the presence of different gp130 epitopes specifically involved in the signaling induced by the cytokines of the gp130 family. In ELISA, only mAb of group B and E were found to inhibit the binding of the IL-6-IL-6R complex to gp130, showing that they identified one or two domains of gp130 involved in its interaction with the IL-6-IL-6R complex. Precise identification of this(ese) epitope(s) would be useful to better understand the mechanisms of the IL-6 signalling.

Research paper thumbnail of Human anti-mouse antibody response to the injection of murine monoclonal antibodies against IL-6

Clinical & Experimental Immunology, 2008

We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and... more We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and six with metastatic renal cell carcinoma (MRCC)) who were treated with B-E8, an IgG1 MoAb against IL-6. Efficiency of the treatment was evidenced by the drop in the serum levels of Creactive protein (CRP), the in vivo production of which is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MoAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgGI isotype; in these two patients B-E8 MoAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment remained efficient in the presence of HAMA. Circulating B-E8 MoAbs were still able to bind to IL-6 and to inhibit IL-6-dependent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA produced against MoAb directed against cellular targets, HAMA against anti-IL-6 MoAb idiotopes led neither to clearance nor to functional inactivation of the injected MoAb. This was further shown by resuming the B-E8 treatment with success in a patient who still had anti-idiotypic HAMA.

Research paper thumbnail of Etude structurale et fonctionnelle du récepteur de l'interleukine-6 : interactions moléculaires dans la médiation du message IL-6

L'interleukine-6 (il-6) est une cytokine multifonctionnelle qui est impliquee comme facteur d... more L'interleukine-6 (il-6) est une cytokine multifonctionnelle qui est impliquee comme facteur de croissance dans les proliferations plasmocytaires benignes et malignes. Sa neutralisation a l'aide d'anticorps monoclonaux anti-il-6, entraine le bloquage du clone tumoral chez des patients atteints de myelome multiple (mm). Dans le but d'explorer la possibilite de bloquer le message il-6 en ciblant son recepteur, nous avons produit sept anticorps monoclonaux specifiques de la chaine gp80 du recepteur de l'interleukine-6 (il-6r). Nous avons recherche la fonction des epitopes cibles par nos anticorps. Les etudes d'inhibition des interactions ligand/recepteur et l'inhibition de la proliferation de lignees dependantes de l'il-6 nous ont montre que deux epitopes sont impliques dans la fixation de la cytokine et qu'un troisieme epitope est implique dans l'interaction des deux chaines du recepteur. L'association d'anticorps de specificite different...

Research paper thumbnail of Tumoral Sample Peripheral blood Pleural effusion Peripheral blood Pleural effusion Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Bone marrow and the percentages of myeloma cells

We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to b... more We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to be obtained from each of 10 tumor samples harvested from 9 patients with extramedullary proliferation. Fresh samples were cultured with interleukin 6 (IL-6) and granulocyte macrophagetolony stimulating factor (GM-CSF) at a high cell density and resulting HMCL growth became progressively dependent on IL-6 alone, no longer requiring GM-CSF. These HMCL, which had the same immunoglobulin gene rearrangements as the patients’ original myeloma cells, were designated XG-l to XG-9. XG HMCL had a plasma cell morphology, expressed plasma cell antigen (Ag), namely cytoplasmic immunoglobulins. CD38, 8-84 Ag, and CD77, and lacked the usual B-cell

Research paper thumbnail of IL-6-induced changes in synthesis of α 1-acid glycoprotein in human hepatoma Hep3B cells are distinctively regulated by monoclonal antibodies directed against different epitopes of IL-6 receptor (gp80)

European cytokine network

ABSTRACT

Research paper thumbnail of Interleukin-6 receptor signaling. I. gp80 and gp130 receptor interaction in the absence of interleukin-6

European cytokine network

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specif... more Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. To study the physiological role of these soluble receptors, both proteins were purified from human plasma and the kinetic constants of equilibria between IL-6 and its natural soluble IL-6R (sIL-6R) and gp130 receptor (sgp130) were measured using surface plasmon resonance analysis. Unexpectedly, natural sIL-6R and natural sgp130 were found to interact (Kd = 2.8 nM) in the absence of IL-6. No interaction was seen between the recombinant soluble receptors or between either natural soluble receptor and its recombinant partner. This binary complex was not due to copurification of IL-6 and was detected in human plasma of healthy donors. It results from either direct interaction between the two natural soluble receptors or indirect binding mediated by a yet unidentified copurified plasma molecule playing the role of an IL-6 antagonist. Once formed, the binary complex was found to be unable to bind IL-6. Soluble gp130 had already been shown to inhibit IL-6 signaling by inactivating the IL-6/IL-6R complex. In addition we show that, in the absence of IL-6, circulating natural sgp130 is able to inhibit directly the circulating sIL-6R that is a strong synergic molecule of IL-6 signaling.

Research paper thumbnail of Gamma-interferon induces apoptosis of the B lymphoma WEHI-279 cell line through a CD95/CD95L-independent mechanism

European cytokine network

Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and ... more Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and natural killer (NK) cells, plays a central role in the development of humoral and cell-mediated immunity. IFN-gamma participates in the maturation and differentiation of B cells, but it has been previously reported that IFN-gamma may inhibit the early stages of B cell activation. We report that the inhibition of the B lymphoma cell WEHI-279-proliferation induced by IFN-gamma, involves the induction of typical features of apoptosis (nuclear chromatin condensation and fragmentation, cell shrinkage, phosphatidyl-serine (PS) exposure and mitochondrial membrane potential (delta psim) loss). IFN-gamma-mediated B cell apoptosis was decreased by the addition of the T helper type 2 cytokine, IL-4. WEHI-279 cells express CD95 and undergo apoptosis after treatment with either an agonistic anti-CD95 Ab or with a soluble recombinant CD95L. However, incubation with CD95-Fc or TRAIL-R1-Fc fusion protei...

Research paper thumbnail of Reproducible obtaining of human myeloma cell lines as a model for tumor stem cell study in human multiple myeloma

Blood, Jan 15, 1994

We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to b... more We report a novel, reproducible methodology which enabled 10 human myeloma cell lines (HMCL) to be obtained from each of 10 tumor samples harvested from 9 patients with extramedullary proliferation. Fresh samples were cultured with interleukin 6 (IL-6) and granulocyte macrophage-colony stimulating factor (GM-CSF) at a high cell density and resulting HMCL growth became progressively dependent on IL-6 alone, no longer requiring GM-CSF. These HMCL, which had the same immunoglobulin gene rearrangements as the patients' original myeloma cells, were designated XG-1 to XG-9. XG HMCL had a plasma cell morphology, expressed plasma cell antigen (Ag), namely cytoplasmic immunoglobulins, CD38, B-B4 Ag, and CD77, and lacked the usual B-cell Ag. They also expressed activation antigens such as CD28 with coexpression of CD28 and its ligand, B7 Ag, in four HMCL. Six HMCL expressed CD40, 4 CD23, and 5 its ligand, CD21. The XG HMCL bore adhesion molecules VLA-4 and CD44 (all 10 HMCL), VLA-5 (7 HMC...

Research paper thumbnail of Immunomodulating IL-6 activity by murine monoclonal antibodies

International journal of immunopharmacology, 1995

The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injec... more The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injection of murine monoclonal antibodies (MAb) directed against cellular targets, has been reported extensively in several studies. We analysed here HAMA in 12 patients (six with multiple myeloma, MM, and six with metastatic renal cell carcinoma, MRCC) who were treated with B-E8, an IgG1 MAb against interleukin-6 (IL-6). Efficiency of the treatment was evidenced by the drop in the serum levels of C reactive protein (CRP), of which the in vivo production is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All of immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgG1 isotype; in these two patients B-E8 MAb cleared rapidly from ...

Research paper thumbnail of Inhibiting IL-6 in human multiple myeloma

Current topics in microbiology and immunology, 1992

It has been well established by several groups that IL-6 is overproduced in patients with Multipl... more It has been well established by several groups that IL-6 is overproduced in patients with Multiple Myeloma (MM) and that this cytokine is an essential growth factor of myeloma cells both in vitro and in vivo (1–5). Most investigators also agree now that IL-6 is produced mainly by bone-marrow stromal cells (1, 6). The aim of this report was to summarize our recent results concerning the mechanisms of IL-6 production control by the bone-marrow environment and the different means of neutralizing IL-6 activity in vitro and in vivo.

Research paper thumbnail of Interleukin-6 receptor signaling. II. Bio-availability of interleukin-6 in serum

European cytokine network, 1999

Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specif... more Interleukin-6 (IL-6) is used as a growth factor by various tumor cells. It binds to a gp80 specific receptor (IL-6R) and then to a gp130 transducing chain. Both receptor chains are released as soluble functional proteins which circulate in biological fluids. With a view to studying the physiological role of these soluble receptors, both proteins were purified from human plasma. Surface plasmon resonance was used to measure the kinetic constants of equilibria between IL-6 and natural sIL-6R, and between the IL-6/sIL-6R complex and soluble gp130. Kd values were found to be 0. 9 and 2.3 nM respectively. Soluble natural IL-6R and gp130 were also found to interact with a Kd of 2.8 nM in the absence of IL-6. By using these Kd values, a mathematical simulation predicted that 1) within a large range of IL-6, sIL-6R and sgp130 concentrations, free IL-6 represents 30% of the total circulating cytokine, 2) sIL-6R overconcentrations lead to dramatic changes of the concentration of free IL-6, 3)...

Research paper thumbnail of Immunomodulating IL6 activity by murine monoclonal antibodies

International Journal of Immunopharmacology, 1995

The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injec... more The human anti-mouse immunoglobulin antibody (HAMA) response, which occurs frequently after injection of murine monoclonal antibodies (MAb) directed against cellular targets, has been reported extensively in several studies. We analysed here HAMA in 12 patients (six with multiple myeloma, MM, and six with metastatic renal cell carcinoma, MRCC) who were treated with B-E8, an IgG1 MAb against interleukin-6 (IL-6). Efficiency

Research paper thumbnail of Functional interaction of the gp80 and gp130IL-6 receptors in human B cell malignancies

Clinical and Laboratory Haematology, 1998

IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggest... more IL-6, or cytokines of the IL-6 family using gp130 as transducer chain receptor, have been suggested to play a role in certain B lymphoid neoplasia. The presence of cell membrane gp80 and gp130 IL-6 receptors was studied in 98 patients with various leukaemia and non-Hodgkin&#39;s malignant lymphoma using flow cytofluorometry and immunohistology. Except neoplasia of immature B cells which expressed neither of the receptors, the majority of B cell tumours expressed one or both of them, mantle cell lymphoma being found to express the highest density of receptors. Using IL-6-dependent XG myeloma cell lines and mAb recognizing various gp80 and gp130 functional epitopes, it has been shown that IL-6 activation leads to a modified expression of some epitopes. In particular, the decrease or the disappearance of a gp130 epitope called A1 signed gp130 dimerization which is the first step of the gp130 activation pathway. Gp80 and gp130 epitope analysis was achieved in 17 of the patients. In four, an epitope phenotype compatible with a cytokine-induced activation was found. The cells of five B-CLL patients which expressed both gp80 and gp130 receptors were incubated with IL-6 to induce activation. In three of the cases they were found to rearrange their receptors in activated forms but not in the two others, showing that cells able to be activated or not can be found. These results confirm that gp130 signalling might play an important role in the pathogenesis of certain B cell neoplasia.

Research paper thumbnail of Dimerization and activation of the common transducing chain (gp130) of the cytokines of the IL-6 family by mAb

International Immunology, 1998

The objective of our research was to study the mechanisms of activation of mAb against the gp130 ... more The objective of our research was to study the mechanisms of activation of mAb against the gp130 transducer chain common to the IL-6 cytokine family. It has been found that among the 56 anti-gp130 available worldwide, none was able to activate the growth of IL-6-dependent myeloma cell lines. When certain of them were associated in pairs they allowed the cells to grow; alone, they were inhibitory. The same activation was also obtained by cross-linking certain anti-gp130 mAb on the cell membrane with a goat anti-mouse Ig antiserum. A bispecific mAb was prepared by the somatic fusion of two hybridomas secreting two mAb whose association was able to activate gp130 signaling; the bispecific mAb was inactive. The activating mAb were able to support long-term proliferation of the IL-6-dependent myeloma cell lines, which indicates that they are potential valuable growth factors of tumor cells and hematopoietic stem cells. When they were injected into SCID mice, they allowed human IL-6dependent myeloma cell lines to grow, develop tumors and metastasize. By studying the functional epitopes of the cell membrane gp130 receptors, it was shown that the activating mAb induced gp130 dimerization and STAT3 activation, as did IL-6.

Research paper thumbnail of Identification of a novel antigenic structure of the human receptor for interleukin-6 involved in the interaction with the glycoprotein 130 chain

Immunology, 1996

The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80)... more The receptor for interleukin-6 (IL-6) is characterized by a ligand-binding glycoprotein 80 (gp80) transmembrane chain (IL-6R) which associates with a signal-transducer gp130 chain. We previously raised a series of monoclonal antibodies (mAb) recognizing different epitopes of the human IL-6R and interfering with the function of the receptor. One of them, M182, was able to diminish the proliferation of IL-6-dependent plasmacytoma cell lines although it was found unable to inhibit the binding of IL-6 to its receptor. Using an enzyme-linked immunosorbent assay for measuring the binding of IL-6-IL-6R to the gpl30 chain, we showed that M182 was directed against a structure directly involved in the IL-6R/gpl 30 interaction. M 182 was able to potentiate the inhibitor effect of anti-IL-6R mAB which interfere with the binding of IL-6, leading to complete inhibition of the proliferation of IL-6-dependent cell lines. M182 was also found to synergize with inhibitory anti-IL-6 mAb. Therefore this structure appears to be an important regulatory domain of the IL-6R and a valuable target for inhibiting IL-6 signalling. INTRODUCTION Cell surface interleukin-6 (IL-6) receptors (IL-6R) belong to a class characterized by a complex hetero-oligomeric structure in which ligand-binding and signal-transduction are compartmentalized into distinct receptor units. Two transmembrane proteins are involved, a glycoprotein (gp)80 ligand-binding receptor (IL-6R) and an associated transducer gpl3O molecule.' Upon binding of IL-6 to its IL-6R, the IL-6-IL-6R complexes associate with the gpl30 molecules, resulting in the formation of high avidity IL-6-binding receptors and activation of transduction signals.' Membrane IL-6R is cleaved from the cell membrane2 or translated from an alternative spliced mRNA3'4 to give a gp55 soluble form (sIL-6R). It was demonstrated that both genetically engineering and plasma sIL-6R retained their capability to bind IL-6 and that the complexes formed were able to activate the gpl30 transducer chain.56 Therefore, in contrast with other soluble cytokine receptors which are generally antagonists,7 sIL-6R are able to promote IL-6 activity. IL-6 has recently been associated with a cell growth function of tumoral plasma cells in multiple myeloma.8'9

Research paper thumbnail of Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy

European Journal of Immunology, 1993

Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patien... more Increased and highly stable levels of functional soluble interleukin-6 receptor in sera of patients with monoclonal gammopathy" Soluble human interleukin-6 receptor (sIL-6R) was measured in the serum of 30 healthy individuals, 32 individuals with monoclonal gammopathy of undetermined significance (MGUS), 20 patients with early multiple myeloma (MM) and 54 patients with overt MM. The serum activity recognized by an immunoradiometric assay was determined to be s I L-~R , because of its binding capacity to IL-6 and its molecular mass of 55 kDa. All sera of healthy individuals contained sIL-6R (mean value: 89 ng/ml, range 17-300 ng/ml). Serum sIL-6R levels were increased by 51 % in patients with MGUS (mean value: 135 ng/ml, p C 0.005), by 44% in patients with early myeloma (mean value: 128 ng/ml, p C 0.001) and by 116 YO in patients with overt MM (mean value: 193 ng/ml,p < 0.001). In patients withMM, acomplete lackof correlation (p > 0.7) was found between serum sIL-6R levels and other previously recognized prognostic factors in this disease, particularly serum IL-6 levels and those factors related to tumor cell mass. The independence of serum sIL-6R levels on tumor cell mass was directly demonstrated by studying four patients with MM treated with autologous bone marrow transplantation for periods of between 320 and 760 days. These levels were found to be remarkably stable and constant, independent of whether patients relapsed or achieved complete remission. Finally, physiological concentrations of sIL-6R were found to increase by tenfold the sensitivity of human myeloma cell lines to L-6.These observations suggest a high control of the sIL-6R level in vivo, and, possibly, an important functional role of this circulating protein in patients with monoclonal gammopathies.

Research paper thumbnail of Identification of centerin: a novel human germinal center B cell-restricted serpin

European Journal of Immunology, 2000

Research paper thumbnail of Optimizing therapeutic strategies to inhibit circulating soluble target molecules with monoclonal antibodies: example of the soluble IL-6 receptors

European Journal of Immunology, 2001

Therapeutic targeting of soluble molecules such as cytokines can be achieved with monoclonal anti... more Therapeutic targeting of soluble molecules such as cytokines can be achieved with monoclonal antibodies (mAb). Anti-IL-6 mAb have been shown to form circulating complexes, resulting in the increase of the half-life of the cytokine in vivo. In IL-6-related diseases, the soluble human IL-6 receptors (shIL-6R), which have been shown to possess strong agonist activity, circulate in the plasma at a high concentration and must be neutralized. Their clearance was studied in mice that had been made to express circulating shIL-6R after i.p. grafting of mouse thymoma cells transfected with a gene coding for shIL-6R, treated with various anti-shIL-6R mAb recognizing different epitopes of the molecule. Injection of one anti-hIL-6R mAb stabilized the short-lived hIL-6R and led to their accumulation. The same result was observed when two mAb directed against two different epitopes of the hIL-6R were used. Clearance of the receptors was only achieved when three mAb specific for three different epitopes were injected. A permanent clearing of the hIL-6R could be obtained by repeated injections of the clearing mixture. No correlation was found between the ability of the mAb to clear the sIL-6R and to immunoprecipitate them in agarose gel. The F(ab') 2 fragments lost the clearing ability of the intact mAb. These results clearly show that therapeutic clearance of sIL-6R by mAb need at least three mAb directed against three different epitopes of the molecule, a conclusion which is likely to apply for clearing any soluble target molecule.

Research paper thumbnail of Activation of the gp130 signaling pathway by monoclonal antibodies directed against the gp130 molecule

European Journal of Immunology, 1997

Six cytokines of the interleukin (IL)-6 family involved in various inflammatory or tumoral diseas... more Six cytokines of the interleukin (IL)-6 family involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. We made a panel of anti-gp130 monoclonal antibodies (mAb) to study the structure and function of the gp130 molecule. These mAb recognized different epitopes of the gp130 that we called A to J. Most of the mAb were found to be inhibitors and we studied whether some of them could also induce gp130 activation. When used alone, none of them was able to initiate the proliferation of IL-6-dependent cell lines. However, some particular associations of the mAb were able to induce a proliferative response. mAb B1 could activate the lines in association with F1 or with I2 but not with I1, which in ELISA was similar to I2. In contrast mAb B2, which in ELISA appeared to be very similar to B1, was able to activate the cells in association with I1 but not with F1 or I2. Two other mAb belonging to specificities A and C were found to be activators either in association with I1 only, or with I1 or B2, respectively. These associations of mAb appeared to be nearly as potent activators as IL-6 itself. Although we still have no precise idea of the mechanisms involved, they are interesting tools to study the molecular interactions leading to gp130 activation and, from a practical point of view, valuable growth factors of hematopoietic stem cells.

Research paper thumbnail of Major role of the soluble interleukin-6/interleukin-6 receptor complex for the proliferation of interleukin-6-dependent human myeloma cell lines

European Journal of Immunology, 1997

Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activi... more Interleukin-6 (IL-6) is a proinflammatory cytokine which possesses a central growth factor activity for certain tumor cells such as plasma cells in multiple myeloma (MM). Upon binding of IL-6, soluble IL-6 receptor (sIL-6R) has been shown to retain its affinity for IL-6 and to associate with the signal-transducing gp130 chain. Therefore, contrary to the majority of soluble cytokine receptors, it plays an agonist role in IL-6 signaling. In order to test its physiological importance as compared to that of its membrane counterpart, we studied cells from two myeloma cell lines which need exogenous IL-6 to proliferate and release sIL-6R into their culture supernatant. Using a new culture system where the supernatant recirculated permanently through an anti-IL-6R affinity column, all sIL-6R was removed from the culture medium throughout the culture period. Under these conditions IL-6-dependent cells were unable to grow in the presence of physiological concentrations of IL-6, showing the major role of the sIL-6R for sustaining the proliferation of these cell lines. Increasing IL-6 concentrations well over the physiological values allowed the cells to proliferate again. No effect was seen when sIL-6R was removed from the supernatant of an IL-6-independent myeloma cell line. These results show that the levels of circulating sIL-6R (and thus those of IL-6/sIL-6R complex) are worth looking at in pathologies involving IL-6 hyperactivity.

Research paper thumbnail of SPECIFIC INHIBITION OF IL-6 SIGNALLING WITH MONOCLONAL ANTIBODIES AGAINST THE gp130 RECEPTOR

Cytokine, 1997

A family of cytokines [IL-6, IL-11, oncostatin M (OM), leukaemia inhibitory factor (LIF), ciliary... more A family of cytokines [IL-6, IL-11, oncostatin M (OM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF) and cardiotrophin-1] involved in various inflammatory or tumoral diseases share the same gp130 signal transducer chain. The complex formed with their specific receptors associates with a common transducing gp130 membrane protein (gp130) resulting in the formation of high avidity receptor and activation of tyrosine kinases. With the view of identifying gp130 domains specifically involved in IL-6 signalling, the authors prepared 37 new anti-gp130 mAb and analysed the structure-function relationship of the molecule. By cross-competition ELISA, the mAb were classified in 10 subgroups called A to J. By ELISA and BIAcore analysis, the mAb were found to recognize at least 18 antigenic specificities of the gp130 chain. The mAb reacted against the soluble and the membrane forms of gp130 as well. Their ability to inhibit the proliferation of the human myeloma cell line XG-4 of which the growth is strictly dependent on the presence of either exogenous IL-6, or LIF, or OM, or CNTF was studied. Besides mAb with no evident neutralizing effect (G and H) and mAb which neutralized equally well the activity of all tested cytokines (all mAb of groups A, I and J), some showed a selective effect. Those of group F inhibited also the proliferation induced by the 4 cytokines, but more specifically that dependent on the CNTF. mAb of groups B and E specifically inhibited the growth induced by IL-6, whereas those of group C inhibited that induced by LIF and OM. These results show the presence of different gp130 epitopes specifically involved in the signaling induced by the cytokines of the gp130 family. In ELISA, only mAb of group B and E were found to inhibit the binding of the IL-6-IL-6R complex to gp130, showing that they identified one or two domains of gp130 involved in its interaction with the IL-6-IL-6R complex. Precise identification of this(ese) epitope(s) would be useful to better understand the mechanisms of the IL-6 signalling.

Research paper thumbnail of Human anti-mouse antibody response to the injection of murine monoclonal antibodies against IL-6

Clinical & Experimental Immunology, 2008

We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and... more We analysed human anti-mouse antibodies (HAMA) in 12 patients (six with multiple myeloma (MM) and six with metastatic renal cell carcinoma (MRCC)) who were treated with B-E8, an IgG1 MoAb against IL-6. Efficiency of the treatment was evidenced by the drop in the serum levels of Creactive protein (CRP), the in vivo production of which is under the control of IL-6. Three patients with MM and the six patients with MRCC became immunized to the injected MoAb. HAMA appeared between days 7 and 15 after the beginning of the treatment. The nine patients made IgG antibodies; four also made IgM. All immunized patients made anti-idiotype antibodies specific to B-E8. Two of them also developed HAMA directed to murine IgGI isotype; in these two patients B-E8 MoAb cleared rapidly from the circulation with loss of treatment efficiency. In the patients who developed only anti-idiotype antibodies, serum levels of B-E8 remained unchanged and CRP production remained inhibited, indicating that treatment remained efficient in the presence of HAMA. Circulating B-E8 MoAbs were still able to bind to IL-6 and to inhibit IL-6-dependent proliferation despite the presence of anti-idiotypic HAMA. Therefore, in contrast to HAMA produced against MoAb directed against cellular targets, HAMA against anti-IL-6 MoAb idiotopes led neither to clearance nor to functional inactivation of the injected MoAb. This was further shown by resuming the B-E8 treatment with success in a patient who still had anti-idiotypic HAMA.