Discovery and characterization of olokizumab: A humanized antibody targeting interleukin-6 and neutralizing gp130-signaling

Interleukin-6 (IL-6) is a critical regulator of the immune system and has been widely implicated in autoimmune disease. Here, we describe the discovery and characterization of olokizumab, a humanized antibody to IL-6. Data from structural biology, cell biology and primate pharmacology demonstrate the therapeutic potential of targeting IL-6 at “Site 3”, blocking the interaction with the signaling co-receptor gp130.     

Regenerating gene I regulates interleukin-6 production in squamous esophageal cancer cells

Regenerating gene (REG) I plays important roles in cancer cell biology. The purpose of this study was to determine whether REG I affects cytokine production in cancer cells. We transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and Iβ and examined its effects on cytokine expression. We found that transfecting TE-5 and TE-9 cells with REG I Iα and Iβ led to significantly increased expression of interleukin (IL)-6 mRNA and protein, but it had little or no effect on expression of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, interferon-γ, tumor necrosis factor-α, granulocyte-colony stimulating factor or transforming growth factor-β1. The elevated IL-6 expression seen in REG Iα transfectants was silenced by small interfering RNA-mediated knockdown. These finding suggest that REG I may act through IL-6 to exert effects on squamous esophageal cancer cell biology.     

Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.     

IL-6 Trans-Signaling via the Soluble IL-6 Receptor: Importance for the Pro-Inflammatory Activities of IL-6

Interleukin-6 (IL-6) is a cytokine with many activities. It has functions in the regulation of the immune system and the nervous system. Furthermore, IL-6 is involved in liver regeneration and in the metabolic control of the body. On target cells, IL-6 binds to an 80 kDa IL-6 receptor (IL-6R). The complex of IL-6 and IL-6R associates with a second protein, gp130, which thereupon dimerizes and initiates intracellular signaling. Whereas gp130 is expressed on all cells, IL-6R is only present on few cells in the body including hepatocytes and some leukocytes. Cells, which do not express IL-6R cannot respond to the cytokine, since gp130 alone has no measurable affinity for IL-6. Interestingly, a soluble form of IL-6R (sIL-6R) comprising the extracellular portion of the receptor can bind IL-6 with a similar affinity as the membrane bound IL-6R. The complex of IL-6 and sIL-6R can bind to gp130 on cells, which do not express the IL-6R, and which are unresponsive to IL-6. This process has been called trans-signaling. Here I will review published evidence that IL-6 trans-signaling is pro-inflammatory whereas classic IL-6 signaling via the membrane bound IL-6R is needed for regenerative or anti-inflammatory activities of the cytokine. Furthermore, the detailed knowledge of IL-6 biology has important consequences for therapeutic strategies aimed at the blockade of the cytokine IL-6.     

Role of INTERLEUKIN-6 in the pathogenesis of multiple myeloma

Multiple myeloma (MM) is a currently incurable disease caused by the proliferation of malignant plasma cells. Although the pathogenesis of the disease still remains unclear, recent research in the biology of MM has produced new insights into the factors that control the growth and survival of myeloma cells. Among the growth factors, interleukin-6 (IL-6) has an essential role. Evidence suggests that IL-6 is not only a growth factor, but also a survival factor in MM, inhibiting apoptosis in myeloma cells. IL-6 interacts with several factors which are involved in the pathogenesis of MM, such as adhesion molecules, tumour suppressor genes and oncogenes. Considering the essential role of IL-6, it could serve as a target for new therapeutic interventions. Neutralizing the effect of IL-6 may result in a regression of tumour progression.     

一种简单快速克隆IL-6信号转导相关基因的方法

细胞因子作用于受体时的一个重要结果是诱导基因表达。为了克隆与IL-6诱导相关的基因,我们利用一个快速的改良DD-PCR方法,分离并检测了IL-6诱导和未诱导的U937细胞的差异表达基因。用三个完全变性的6—mer引物进行反转录,用2或3个较长的随机引物进行PCR扩增,扩增产物很在2%琼脂糖凝胶电泳上分离,之后回收差异片段并直接用于克隆和测序。在研究中,获得了7个不同的EST,序列分析表明其中2个EST可能是与细胞信号转导相关的新基因片段;反向Northern杂交证实它们是与IL-6作用相关的差异表达基因。     

The Soluble Interleukin-6 Receptor Is a Mediator of Hematopoietic and Skeletal Actions of Parathyroid Hormone

Both PTH and IL-6 signaling play pivotal roles in hematopoiesis and skeletal biology, but their interdependence is unclear. The purpose of this study was to evaluate the effect of IL-6 and soluble IL-6 receptor (sIL-6R) on hematopoietic and skeletal actions of PTH. In the bone microenvironment, PTH stimulated sIL-6R protein levels in primary osteoblast cultures in vitro and bone marrow in vivo in both IL-6^+/+ and IL-6^−/− mice. PTH-mediated hematopoietic cell expansion was attenuated in IL-6^−/− compared with IL-6^+/+ bone marrow, whereas sIL-6R treatment amplified PTH actions in IL-6^−/− earlier than IL-6^+/+ marrow cultures. Blocking sIL-6R signaling with sgp130 (soluble glycoprotein 130 receptor) inhibited PTH-dependent hematopoietic cell expansion in IL-6^−/− marrow. In the skeletal system, although intermittent PTH administration to IL-6^+/+ and IL-6^−/− mice resulted in similar anabolic actions, blocking sIL-6R significantly attenuated PTH anabolic actions. sIL-6R showed no direct effects on osteoblast proliferation or differentiation in vitro; however, it up-regulated myeloid cell expansion and production of the mesenchymal stem cell recruiting agent, TGF-β1 in the bone marrow microenvironment. Collectively, sIL-6R demonstrated orphan function and mediated PTH anabolic actions in bone in association with support of myeloid lineage cells in the hematopoietic system.     

人IL-6 /sIL-6R 结合分子模型的建立及在药物筛选中的应用

目的：建立人IL-6 /sIL-6R 结合的分子模型,用于筛选IL-6 /sIL-6R的抑制剂。方法：将人IL-6基因克隆至原核表达载体pET28a（+）中表达IL-6蛋白,western blot及人IL-6检测试剂盒分析鉴定表达蛋白。同法将人sIL-6R在pET15b载体中表达,纯化并用western blot检测目的蛋白。依据ELISA原理建立IL-6 /sIL-6R 结合的分子模型,并通过改变IL-6、sIL-6R及IL-6 antibody的浓度来优化该模型,用于IL-6 /sIL-6R拮抗药物的筛选。结果：人IL-6可在载体PET28a（+）中高效表达,且经western blot鉴定正确,人IL-6检测试剂盒检测显示具有较高的免疫活性。sIL-6R在PET15b中表达,western blot鉴定正确。通过对IL-6 /sIL-6R结合的分子模型的优化,得到其最佳条件为：IL-6R 1?g/well, IL-6 500ng/well, IL-6 antibody 1?g/well。应用该模型筛选发现有些化合物可显著抑制IL-6与其受体的结合。结论：成功构建IL-6 /sIL-6R结合的分子模型,为高通量筛选IL-6拮抗剂提供平台。     

白介素6的信号转导及其相关疾病的治疗策略

IL-6是一种多功能的细胞因子, 同时IL-6的过度表达与一些疾病的发生和发展有密切关系.IL-6通过一个双链受体系统作用于靶细胞.实验结果表明,IL-6与80 ku的配基结合链IL-6R和信号转导子gp130构成一个相互作用的异六聚体模式,通过gp130的二聚体化引发胞内的信号转导.IL-6胞内的信号转导途径有两种:Jak-STATs路径和Ras-MAPK级联反应路径.靶向IL-6信号转导的药物设计和筛选研究将为IL-6相关疾病的治疗奠定坚实的基础.     

变形链球菌细胞壁对EAhy926细胞TLR4、IL-6和IL-8表达的影响

【目的】观察变形链球菌细胞壁对EAhy926细胞的增殖活性、TLR4的表达和炎性细胞因子IL-6和IL-8分泌的影响,初步探讨变形链球菌致血管内皮细胞TLR4表达、炎症反应及其两者之间的关系。【方法】变形链球菌细胞壁作用于EAhy926细胞,MTT法检测EAhy926细胞的增殖活性;RT-PCR法检测EAhy926细胞TLR4、IL-6和IL-8 mRNA的表达;流式细胞术检测EAhy926细胞表面TLR4的表达;细胞生物活性方法和ELISA分别检测EAhy926细胞IL-6和IL-8的分泌;抗体阻断实验观察IL-6和IL-8的表达与TLR4的关系。【结果】不同浓度的变形链球菌细胞壁作用6 h可促进EAhy926细胞的增殖(P0.05),但12 h后可明显抑制该细胞的生长(P0.05),呈现显著的时间和剂量依赖性。变形链球菌细胞壁作用于EAhy926细胞后,TLR4 mRNA和蛋白水平的表达量随着作用时间延长而逐渐增高,在16 h达到高峰,24 h后又逐渐下降(P0.01);IL-6和IL-8的表达也呈现明显的时间依赖性增高(P0.05)。经TLR4抗体阻断后,变形链球菌细胞壁刺激EAhy926细胞IL-6和IL-8的产生明显减少(P0.01)。【结论】变形链球菌细胞壁可明显抑制EAhy926细胞的生长,上调该细胞TLR4的表达,促进炎性细胞因子IL-6和IL-8的分泌;IL-6和IL-8的产生与TLR4的表达上调密切相关。     

靶向人白介素-6蛋白的治疗性单克隆抗体研究进展*

白介素-6(interleukin-6,IL-6)作为一种多效的细胞因子,参与机体内众多生理与病理过程。研究表明,IL-6首先与自身受体(IL-6R、gp130)形成异源六聚体复合物,进而激活下游信号转导通路,最终发挥生物学功能。 IL-6信号通路异常活化及功能失调与多种疾病密切相关,如自身免疫疾病、慢性炎症、恶性肿瘤等。另外IL-6的异常表达在新型冠状病毒肺炎(COVID-19)细胞因子风暴综合征(CSS)中也扮演重要角色。一般而言,阻断IL-6信号通路上的各关键节点,均可用于IL-6相关疾病的治疗。有别于阻断IL-6R或gp130等公共受体分子,阻断IL-6蛋白的治疗性单克隆抗体特异性更高,在临床研究中,部分品种显示出其独有的治疗特点及有益的疗效。现阶段只有1个靶向IL-6蛋白的单克隆抗体药物获美国FDA批准上市,以及超过8个治疗性单克隆抗体在临床研究阶段。重点对国内外靶向人IL-6蛋白的治疗性单克隆抗体及其临床应用进行综述。     

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles

Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.     

The pyrimidinergic P2Y6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with UDP

The human P2Y6 receptor (hP2Y6) is a member of the G protein-coupled pyrimidinergic P2 receptor family that responds specifically to the extracellular nucleotide uridine diphosphate (UDP). Recently, the hP2Y6 receptor has been reported to mediate monocyte IL-8 production in response to UDP or lipopolysaccharide (LPS), but the role of hP2Y6 in regulating other pro-inflammatory cytokines or mediators is largely unknown. We demonstrate here that UDP specifically induces soluble TNF-alpha and IL-8 production in a promonocytic U937 cell line stably transfected with hP2Y6. However, we did not detect IL-1alpha, IL-1beta, IL-6, IL-10, IL-18, and PGE2 in the conditioned media from the same cell line. These results distinguish UDP/P2Y6 signaling from LPS signaling. Interestingly, UDP induces the production of IL-8, but not TNF-alpha, in human astrocytoma 1321N1 cell lines stably transfected with hP2Y6. Therefore, the immune effect of UDP/P2Y6 signaling on the production of proinflammatory cytokines is selective and dependent on cell types. We further identify that UDP can also induce the production of proinflammatory chemokines MCP-1 and IP-10 in hP2Y6 transfected promonocytic U937 cell lines, but not astrocytoma 1321N1 cell lines stably transfected with hP2Y6. From the Taqman analysis, UDP stimulation significantly upregulates the mRNA levels of IL-8, IP-10, and IL-1beta, but not TNF-alpha. Taken together, these new findings expand the pro-inflammatory biology of UDP mediated by the P2Y6 receptor.     

Differential involvement of central and peripheral norepinephrine in the central lipopolysaccharide-induced interleukin-6 responses in mice

Intracerebroventricular injection of lipopolysaccharide (LPS) induces a marked increase in circulating interleukin (IL)-6 levels and in IL-6 mRNA expression in brain and peripheral organs. Recently, it was reported that intraperitoneal administration of alpha-adrenoceptor antagonists inhibits centrally injected LPS-induced increases in plasma IL-6 levels, suggesting the involvement of the norepinephrine (NE) system in the central LPS-induced IL-6 response. However, the localization (either central or peripheral) of NE involvement in the central LPS-induced IL-6 response has not been characterized. In the present study, mice were pretreated with 6-hydroxydopamine (6-OHDA) administered intracerebroventricularly or intraperitoneally to deplete central or peripheral stores of NE, respectively. Intracerebroventricular LPS (50 ng/mouse) markedly increased plasma IL-6 levels and IL-6 mRNA expression in choroid plexus, hypothalamus, pituitary, adrenals, heart, liver, spleen, and lymph nodes, but with minimal effect in lung, kidney, and testis, as revealed by RT-PCR. Pretreatment with intracerebroventricular 6-OHDA (50 microg/mouse) decreased the LPS-induced plasma IL-6 levels by 39% and the LPS-induced IL-6 mRNA expression in liver, spleen, and lymph nodes, but not in choroid plexus, hypothalamus, pituitary, adrenals, and heart. Pretreatment with intraperitoneal 6-OHDA (100 mg/kg) decreased the LPS-induced plasma IL-6 levels by 36% and the LPS-induced IL-6 mRNA expression in all the peripheral organs displaying increased IL-6 mRNA. Central LPS-induced increase in plasma corticosterone levels was decreased slightly by central but not by peripheral NE depletion. These results suggest that central NE and peripheral NE are differentially involved in the central LPS-induced IL-6 mRNA expression in peripheral organs.     

Cellular senescence or EGFR signaling induces Interleukin 6 (IL-6) receptor expression controlled by mammalian target of rapamycin (mTOR)

Interleukin 6 (IL-6) signaling plays a role in inflammation, cancer, and senescence. Here, we identified soluble IL-6 receptor (sIL-6R) as a member of the senescence-associated secretory phenotype (SASP). Senescence-associated sIL-6R upregulation was mediated by mammalian target of rapamycin (mTOR). sIL-6R was mainly generated by a disintegrin and metalloprotease 10 (ADAM10)-dependent ectodomain shedding to enable IL-6 trans-signaling. In vivo, heterozygous PTEN-knockout mice exhibited higher mTOR activity and increased sIL-6R levels. Moreover, aberrant EGF receptor (EGFR) activation triggered IL-6 synthesis. In analogy to senescence, EGFR-induced activation of mTOR also induced IL-6R expression and sIL-6R generation. Hence, mTOR activation reprograms IL-6 non-responder cells into IL-6 responder cells. Our data suggest that mTOR serves as a central molecular switch to facilitate cellular IL-6 classic and trans-signaling via IL-6R upregulation with direct implications for cellular senescence and tumor development.     

多发性骨髓瘤与白细胞介素-6

白细胞介素-6是多发性骨髓瘤(multiple myeloma,MM)瘤细胞生长和生存的关键因子,也是MM患者发病的主要因子。IL-6/IL-6R系统通过不同通路影响多发性骨髓瘤瘤细胞生长并导致患者骨损害、类风湿性关节炎等一系列并发症。针对IL-6/IL-6R系统的治疗方法将给MM的治疗带来重大的进展。     

慢病毒载体介导shRNA干扰IL-6Rα基因表达削弱IL-6对猪脂肪细胞分化的影响

为研究白细胞介素-6(IL-6)对猪脂肪细胞分化的影响及其分子机制,构建猪IL 6Rα基因RNA干扰（RNA interference, RNAi）慢病毒载体;用IL-6Rα-RNAi重组慢病毒预处理原代培养的猪前体脂肪细胞或不处理, 然后用100 ng/mL IL-6处理分化第6 d的脂肪细胞48 h．通过测定甘油释放量检测脂肪细胞的脂解率;油红O染色提取法测定脂肪细胞的脂质含量;采用RT-PCR 和Western印迹检测脂肪细胞分化相关基因的mRNA 和蛋白表达．结果显示,IL-6显著抑制猪脂肪细胞分化,并下调PPARγ2、Perilipin A和IRS-1的mRNA及蛋白表达,同时增强ERK1/2磷酸化;IL-6Rα-RNAi预处理前体脂肪细胞则显著逆转IL 6的上述作用．总之,IL-6通过多重机制抑制猪脂肪细胞分化;而且本研究构建的IL-6Rα-RNAi重组慢病毒载体可有效阻断IL-6信号,为进一步研究IL-6的功能奠定了基础．     

Gp130 activation by soluble interleukin-6 receptor/interleukin-6 enhances osteoblastic differentiation of human bone marrow-derived mesenchymal stem cells

Interleukin-6 (IL-6) promotes osteodifferentiation in bone-located progenitors; however, it is not known whether this cytokine affects the differentiation of bone marrow-located osteoprogenitors. To address this issue, we prepared human bone marrow-derived mesenchymal stem cells (MSCs), which were characterized by a cell surface phenotype and multipotential nature. It was observed that in the presence of IL-6, MSCs were not differentiated into the osteogenic lineage, as evidenced by a failure to induce alkaline phosphatase activity, an earlier marker of osteodifferentiation. The lack of effect of IL-6 correlates with the observation that MSCs do not express a membrane-bound or soluble IL-6 receptor (sIL-6R). The incompetence of IL-6 was not reversed by the addition of sIL-6R alone or the sIL-6R/IL-6 complex, as it occurs in other IL-6R-negative cells. However, after MSC osteocommittment by dexamethasone, sIL-6R or the sIL-6R/IL-6 complex enhanced alkaline phosphatase activity. The effect of sIL-6R or sIL-6R/IL-6 proved to be dependent on gp130 availability, which is expressed by MSCs, and involves stat-3 phosphorylation. These data suggest that IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.