Rocco Savino

Constitutive Activation of Stat3 Signaling Confers Resistance to Apoptosis in Human U266 Myeloma Cells

Interleukin 6 (IL-6) is the major survival factor for myeloma tumor cells and induces signaling through the STAT proteins. We report that one STAT family member, Stat3, is constitutively activated in bone marrow mononuclear cells from patients with multiple myeloma and in the IL-6-dependent human myeloma cell line U266. Moreover, U266 cells are inherently resistant to Fas-mediated apoptosis and express high levels of the antiapoptotic protein Bcl-xL. Blocking IL-6 receptor signaling from Janus kinases to the Stat3 protein inhibits Bcl-xL expression and induces apoptosis, demonstrating that Stat3 signaling is essential for the survival of myeloma tumor cells. These findings provide evidence that constitutively activated Stat3 signaling contributes to the pathogenesis of multiple myeloma by preventing apoptosis.

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL‐6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution‐phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL‐6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL‐6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL‐6 variants complexed with IL‐6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL‐6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL‐6, two IL‐6R alpha and two gp130 molecules. We propose here a model representing the IL‐6 receptor complex as hexameric, which might be common to other helical cytokines.

Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation

Interleukin‐6 (IL‐6) drives the sequential assembly of a receptor complex formed by the IL‐6 receptor (IL‐6R alpha) and the signal transducing subunit, gp130. A model of human IL‐6 (hIL‐6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL‐6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL‐6 and gp130 and therefore essential for IL‐6 signal transduction, were subjected to site‐directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL‐6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild‐type affinity towards hIL‐6‐R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild‐type IL‐6.

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin‐6 (IL‐6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post‐menopausal osteoporosis. IL‐6, a four‐helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low‐affinity IL‐6 receptor (IL‐6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL‐6R alpha. These mutants behave as full and selective IL‐6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL‐6 super‐antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL‐6R alpha. The IL‐6 super‐antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild‐type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL‐6 suggests that it could be possible to design super‐antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver

Studies with tumor necrosis factor p55 receptor‐ and interleukin‐6 (IL‐6)‐deficient mice have shown that IL‐6 is required for hepatocyte proliferation and reconstitution of the liver mass after partial hepatectomy. The biological activities of IL‐6 are potentiated when this cytokine binds soluble forms of its specific receptor subunit (sIL‐6R) and the resulting complex interacts with the transmembrane signaling chain gp130. We show here that double transgenic mice expressing high levels of both human IL‐6 and sIL‐6R under the control of liver‐specific promoters spontaneously develop nodules of hepatocellular hyperplasia around periportal spaces and present signs of sustained hepatocyte proliferation. The resulting picture is identical to that of human nodular regenerative hyperplasia, a condition frequently associated with immunological and myeloproliferative disorders. In high expressors, hyperplastic lesions progress with time into discrete liver adenomas. These data strongly suggest that the IL‐6/sIL‐6R complex is both a primary stimulus to hepatocyte proliferation and a pathogenic factor of hepatocellular transformation.

IL-6 mediates the effects of IL-1 or TNF, but not PTHrP or 1,25(OH)2D3, on osteoclast-like cell formation in normal human bone marrow cultures.

A potent interleukin‐6 (IL‐6) antagonist (Sant 5), which binds tightly to the IL‐6α receptor but has impaired gp130 heterodimerization, has been developed recently by site‐directed mutagenesis of human IL‐6. We report here that Sant 5 inhibits IL‐6‐stimulated osteoclast‐like multinucleated cell (MNC) formation in human marrow cultures but also inhibits the stimulatory effects of IL‐1 or tumor necrosis factor alpha (TNF‐α in these cultures. We further show that a neutralizing antibody to IL‐6 also inhibits the stimulatory effects of IL‐1 or TNF‐α in these cultures. In contrast, Sant 5 had no effect on parathyroid hormone related protein (PTHrP) or 1,25‐dihydroxyvitamin D3 stimulated MNC formation in human marrow cultures. Transfection of a human marrow stromal cell line, which normally induces osteoclast formation through production of IL‐6, with the Sant 5 cDNA driven by a cytomegalovirus (CMV) promoter blocked the capacity of these cells to stimulate osteoclast‐like cell formation. These Sant 5 transfected cells and conditioned media from these cells also inhibited the stimulatory effects of the parent cell line on MNC formation. These data suggest that IL‐6 mediates the effects of IL‐1 and TNF on human osteoclast formation, but in contrast to murine systems, does not mediate the effects of PTHrP. These data further demonstrate that stromal cells transfected with the Sant 5 cDNA can constitutively produce high levels of the IL‐6 antagonist and inhibit osteoclast formation in vitro.

Prolonged Expression and Effective Readministration of Erythropoietin Delivered with a Fully Deleted Adenoviral Vector

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance—i.e., 100% of treated animals showing sustained expression after 4 months—was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

The IL-6 receptor antagonist SANT-7 overcomes bone marrow stromal cell-mediated drug resistance of multiple myeloma cells.

The bone marrow micro‐environment produces a number of different survival factors that are important for the malignant growth and drug resistance of multiple myeloma (MM) cells. One of the main factors reported to be essential for survival and growth of MM cells in some experimental systems is IL‐6. Therefore, the development and testing of substances that interfere with IL‐6 or IL‐6 receptor (IL‐6R) function might have therapeutic value for the treatment of MM. We analyzed the effect of the IL‐6R antagonist SANT‐7 on growth and survival of the IL‐6–dependent MM cell lines INA‐6 and XG‐1 as well as primary MM cells from 7 patients co‐cultured with bone marrow stromal cells (BMSCs). In particular, we were interested in whether SANT‐7 enhances the growth‐inhibitory effects of dexamethasone (Dex) and all‐trans‐retinoic acid (ATRA). None of the drugs when tested as a single substance, including SANT‐7, induced major growth inhibition if MM cells were co‐cultured with primary human BMSCs. However, when Dex and ATRA were given in combination with SANT‐7, strong growth inhibition was achieved in cell lines and primary MM cells. This effect was due to cell‐cycle arrest and induction of apoptosis.

In Vivo Neutralization of Human IL-6 (hIL-6) Achieved by Immunization of hIL-6-Transgenic Mice with a hIL-6 Receptor Antagonist

Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.