Effects of IL-6/IL-6R axis alterations in serum, plasma and cerebrospinal fluid with the schizophrenia: an updated review and meta-analysis of 58 studies.

Studies investigating the association between IL-6/IL-6R axis and schizophrenia (SZ) susceptibility found inconsistent data. To reconcile the results, a systematic review followed by a meta-analysis was performed to assess the associations. This study followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. A comprehensive search of the literature was carried out in July 2022 using electronic databases PubMed, EBSCO, Science Direct, PsychInfo, and Scopus. Study quality was assessed by the Newcastle-Ottawa scale. Pooled standard mean difference (SMD) with 95% confidence interval (CI) was calculated by fixed-effect or random-effect model analysis. Fifty-eight studies were identified, including 4,200 SZ patients and 4,531 controls. Our meta-analysis results showed an increase of IL-6 levels in plasma, serum, or CSF and decreased IL-6R levels in serum in patients under treatment. Further studies are needed to better elucidate the correlation between the IL-6/IL-6R axis and the schizophrenia.

Discovery and characterization of a monoclonal antibody targeting a conformational epitope of IL-6/IL-6Rα to inhibit IL-6/ IL-6Rα/gp130 hexameric signaling complex formation.

The functional interleukin 6 (IL-6) signaling complex is a hexameric structure composed of IL-6, IL-6Rα, and the signaling receptor gp130. There are three different modes of IL-6 signaling, classic signaling, trans-signaling, and trans-presentation, which are not functionally redundant and mediate pleiotropic effects on both physiological and pathophysiological states. Monoclonal antibodies against IL-6 or IL-6Rα have been successfully developed for clinical application. However, designing therapeutic interventions that block specific modes of IL-6 signaling in a pathologically relevant manner remains a great challenge. Here, we constructed a fusion protein Hyper-IL-6 (HyIL-6) composed of human IL-6 and IL-6Rα to develop specific blocking antibodies against the IL-6/IL-6Rα complex. We successfully screened the monoclonal antibody C14mab, which can bind to HyIL-6 with the binding constant 2.86 × 10-10 and significantly inhibit IL-6/IL-6Rα/gp130 complex formation. In vitro, C14mab effectively inhibited HyIL-6-stimulated signal transducer and activator of transcription 3 (STAT3) activation and related vascular endothelial growth factor (VEGF) induction. Moreover, C14mab efficaciously suppressed HyIL-6-induced acute phase response in vivo. Our data from hydrogen-deuterium exchange mass spectrometry demonstrate that C14mab mainly binds to site IIIa of IL-6 and blocks the final step in the interaction between gp130 and IL-6/IL-6Rα complex. Additionally, data from enzyme-linked immunosorbent assays and kinetics assays indicate that C14mab interacts simultaneously with IL-6 and IL-6Rα, while it does not interact with IL-6Rα alone. The unique features of C14mab may offer a novel alternative for IL-6 blockade and illuminate a better therapeutic intervention targeting IL-6.

Blocking only the bad side of IL-6 in inflammation and cancer.

Interleukin-6 (IL-6) is considered an inflammatory cytokine, which is involved not only in most inflammatory states but it also plays a prominent role in inflammation associated cancers. The response of cells to the cytokine strictly depends on the presence of the IL-6 receptor (IL-6R),which presents IL-6 to the signal transducing receptor subunit gp130, which is expressed on all cells of the body. The expression of IL-6R is limited to some cells, which are therefore IL-6 target cells. The IL-6R can be cleaved by proteases and the thus generated soluble IL-6R (sIL-6R) still binds the ligand IL-6. The complex of IL-6 and sIL-6R can bind to gp130 on any cell, induce dimerization of gp130 and intracellular signaling. This process has been named IL-6 trans-signaling. A fusion protein of soluble gp130 with the constant portion of human IgG1 (sgp130Fc) turned out to be a potent and specific inhibitor of IL-6 trans-signaling. In many animal models of human diseases the significance of IL-6 trans-signaling has been analyzed. It turned out that the activities of IL-6 mediated by the sIL-6R are the pro-inflammatory activities of the cytokine whereas activities of IL-6 mediated by the membrane-bound IL-6R are rather protective and regenerative. The sgp130Fc protein has recently been developed into a biologic. The possible consequences of a specific IL-6 trans-signaling blockade is discussed in the light of the recent successfully concluded phase II clinical trials in patients with inflammatory bowel disease.

Molecular dynamics analysis of the binding of human interleukin-6 with interleukin-6 α-receptor.

Human interleukin-6 (hIL-6) is a multifunctional cytokine that regulates immune and inflammatory responses in addition to metabolic and regenerative processes and cancer. hIL-6 binding to the IL-6 receptor (IL-6Rα) induces homodimerization and recruitment of the glycoprotein (gp130) to form a hexameric signaling complex. Anti-IL-6 and IL-6R antibodies are clinically approved inhibitors of IL-6 signaling pathway for treating rheumatoid arthritis and Castleman's disease, respectively. There is a potential to develop novel small molecule IL-6 antagonists derived from understanding the structural basis for IL-6/IL-6Rα interactions. Here, we combine homology modeling with extensive molecular dynamics (MD) simulations to examine the association of hIL-6 with IL-6Rα. A comparison with MD of apo hIL-6 reveals that the binding of hIL-6 to IL-6Rα induces structural and dynamic rearrangements in the AB loop region of hIL-6, disrupting intraprotein contacts and increasing the flexibility of residues 48 to 58 of the AB loop. In contrast, due to the involvement of residues 59 to 78 in forming contacts with the receptor, these residues of the AB loop are observed to rigidify in the presence of the receptor. The binary complex is primarily stabilized by two pairs of salt bridges, Arg181 (hIL-6)- Glu182 (IL-6Rα) and Arg184 (hIL-6)- Glu183 (IL-6Rα) as well as hydrophobic and aromatic stacking interactions mediated essentially by Phe residues in both proteins. An interplay of electrostatic, hydrophobic, hydrogen bonding, and aromatic stacking interactions facilitates the formation of the hIL-6/IL-6Rα complex.

In vitro display evolution of IL-6R-binding unnatural peptides ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid.

The interaction of the multifunctional cytokine interleukin (IL)-6 and its receptor (IL-6R) is involved in various diseases, including not only autoimmune diseases such as rheumatoid arthritis but also cancer and cytokine storms in coronavirus disease 2019 (COVID-19). In this study, systematic evolution of ligands by exponential enrichment (SELEX) against human IL-6R from mRNA-displayed unnatural peptide library ribosomally initiated and cyclized with m-(chloromethyl)benzoic acid (mClPh) incorporated by genetic code expansion (sense suppression) was performed using the PURE (Protein synthesis Using Recombinant Elements) system. A novel 13-mer unnatural mClPh-cyclized peptide that binds to the extracellular domain of IL-6R was discovered from an extremely diverse random peptide library. In vitro affinity maturation of IL-6R-binding unnatural mClPh-cyclized peptide from focused libraries was performed, identifying two IL-6R-binding unnatural mClPh-cyclized peptides by next-generation sequencing. Because cyclization can increase the protease resistance of peptides, novel IL-6R-binding mClPh-cyclized peptides discovered in this study have the potential to be used for a variety of research, therapeutic, and diagnostic applications involving IL-6/IL-6R signaling.

Distance dependent shedding of IL-6R.

Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process. This finding contributes to the understanding of the complex and multilayered regulation of ADAM17 at the cell surface.

I6P7 peptide modified superparamagnetic iron oxide nanoparticles for magnetic resonance imaging detection of low-grade brain gliomas.

Glioma, the most severe primary brain malignancy, has very low survival rates and a high level of recurrence. Nowadays, conventional treatments for these patients are suffering a similar plight owing to the distinctive features of the malignant gliomas, for example chemotherapy is limited by the blood-brain barrier while surgery and radiation therapy are affected by the unclear boundaries of tumor from normal tissue. In the present study, a novel superparamagnetic iron oxide (SPIO) nanoprobe for enhanced T2-weighted magnetic resonance imaging (MRI) was developed. A frequently used MRI probe, SPIO nanoparticles, was coated with a silica outer layer and for the first time was covalently modified with interleukin-6 receptor targeting peptides (I6P7) to promote transportation through the blood-brain barrier and recognition of low-grade gliomas. The efficiency of transcytosis across the blood-brain barrier was examined in vitro using a transwell invasion model and in vivo in nude mice with orthotopic low-grade gliomas. The targeting nanoprobe showed significant MRI enhancement and has potential for use in the diagnosis of low-grade gliomas.

Molecular characterization of grass carp interleukin-6 receptor and the agonistic activity of its soluble form in head kidney leucocytes.

Interleukin (IL)-6 receptor (IL-6R) can specifically bind to IL-6 and the complex subsequently recruits a transmembrane signal transducer, gp130, to trigger the intracellular signal transduction. IL-6R exists in two forms, a transmembrane IL-6R and a soluble IL-6R (sIL-6R), leading to different signal transduction mechanisms as classic signaling and trans-signaling, respectively. There is now a general consensus that these two modes of signal transduction can mediate anti-inflammatory and pro-inflammatory activities of IL-6. The study on Il-6r is limited although Il-6 has been well studied in teleost. In the present study, a cDNA encoding grass carp Il-6r (gcIl-6r) was isolated. An in-silico analysis showed that gcIl-6r shared the same functional domains and conserved gene synteny at its loci with mouse homologue, and its amino acid sequence was conserved in fish species. A tissue distribution assay demonstrated that gcil6r mRNA was expressed with high levels in immune tissues including spleen and head kidney, and its expression was induced by LPS and Poly I:C in grass carp head kidney leucocytes (HKLs). An in vitro binding assay showed that recombinant soluble gcIl-6r (rgcsIl-6r) could specifically bind to recombinant gcIl-6 (rgcIl-6) protein. Moreover, rgcIl-6 stimulated suppressor of cytokine signaling 3 (socs3)'s mRNA expression in grass carp HKLs and it combined with rgcsIl-6r increased socs3 mRNA expression in CIK cells with gp130 but without Il-6r expression. In HKLs, rgcIl-6 stimulated the mRNA levels of both pro-inflammatory (tnfa and il1b) and anti-inflammatory (il10) cytokines, and rgcsIl-6r could augment these stimulatory effects of gcIl-6. Taken these data together, gcsIl-6r can mediate the immuno-regulatory functions of gcIl-6 and has an agonistic property in these actions of Il-6 in grass carp.

Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites.

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman's disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients' blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL-6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.

Molecular Dynamics Simulations Reveal Key Roles of the Interleukin-6 Alpha Receptor in the Assembly of the Human Interleukin-6 Receptor Complex.

Human interleukin-6 (hIL-6) is a pleiotropic cytokine with three distinct receptor epitopes, termed sites I, II, and III, which function to assemble a signaling complex. hIL-6 signals via a glycoprotein 130 (gp130) homodimer after initially forming a heterodimer with the nonsignaling α-receptor (IL-6Rα). The molecular description of the assembly of the hIL-6 signaling complex remains elusive because available structures provide descriptions of hIL-6 in its free and fully bound receptor forms, but not for intermediate steps that are crucial in the stepwise assembly of the signaling complex. In this report, molecular dynamics simulations provide atomic details describing the functional role of the initial hIL-6/IL-6Rα complex in facilitating subsequent interactions with gp130, which have not been previously shown. IL-6Rα binding to hIL-6 rigidifies the flexible N-terminus of the hIL-6 AB-loop through interactions with the D2 domain of IL-6Rα. This rigidification combined with repositioning of residues involved in gp130 receptor recognition promotes gp130 binding at site III. Binding of gp130 receptors at sites II and III is coupled with the release of the hIL-6 N-terminal AB-loop interaction and a pivoting of IL-6Rα around the hIL-6 helix bundle to the state of the hIL-6/IL-6Rα/gp130 complex.

Orthogonal Mass Spectrometry-Based Footprinting for Epitope Mapping and Structural Characterization: The IL-6 Receptor upon Binding of Protein Therapeutics.

Higher-order structure (HOS) is a crucial determinant for the biological functions and quality attributes of protein therapeutics. Mass spectrometry (MS)-based protein footprinting approaches play an important role in elucidating the relationship between protein biophysical properties and structure. Here, we describe the use of a combined method including hydrogen-deuterium exchange (HDX), fast photochemical oxidation of proteins (FPOP), and site-specific carboxyl group footprinting to investigate the HOS of protein and protein complexes. The work focuses on implementing complementary solution-phase footprinting approaches that differ in time scale, specificity for protein residue side chains vs backbone as well as selectivity for different residue types to map integratively the epitope of human interleukin-6 receptor (IL-6R) for two adnectins with distinct affinities (Kd, Adnectin1 ∼ 6.2 pM vs Kd, Adnectin2 ∼ 46 nM). Furthermore, the study evaluates the resultant conformation/dynamic change of IL-6R. The suggested epitope, which is conserved for adnectin1 and adnectin2 binding, is a flexible loop that connects two ß-strands in the cytokine-binding domain (DII) of IL-6R. We also found that adnectin1, the more strongly binding ligand, induces structural perturbations on two unstructured loops that are distally located beyond the epitope. Those changes are either attenuated or not detected for the case of adnectin2 binding. In addition to providing credibility in epitope determination, utilization of those combined approaches reveals the structural effects that can differentiate protein therapeutics with apparently similar biophysical properties.

SorLA in Interleukin-6 Signaling and Turnover.

Interleukin-6 (IL-6) is a multifunctional cytokine with important functions in various physiologic processes. Mice lacking IL-6 exhibit multiple phenotypic abnormalities, such as an inadequate immune and acute-phase response, and elevated levels of circulating IL-6 have been found to accompany several pathological conditions. IL-6 binds the nonsignaling IL-6 receptor (IL-6R), which is expressed as a transmembrane, as well as a secreted circulating protein, before it engages homodimeric gp130 for signaling. Complex formation between IL-6 and the membrane-bound IL-6 receptor gives rise to classic cis signaling, whereas complex formation between IL-6 and the soluble IL-6R results in trans signaling. Here, we report that the endocytic receptor SorLA targets IL-6 and IL-6R. We present evidence that SorLA mediates efficient cellular uptake of both IL-6 and the circulating IL-6R in astrocytes. We further show that SorLA interacts with the membrane-bound IL-6R at the cell surface and thereby downregulates IL-6 cis signaling. Finally, we find that the SorLA ectodomain, released from the cell membrane upon enzymatic cleavage of full-length SorLA, may act as an IL-6 carrier protein that stabilizes IL-6 and its capacity for trans signaling.

Trans-signaling of interleukin-6 (IL-6) is mediated by the soluble IL-6 receptor, but not by soluble CD5.

IL-6 exerts its pleiotropic activities on its target cells via the IL-6 alpha-receptor (IL-6R), which is expressed on a limited number of cell types. IL-6 can further signal via soluble forms of its receptor (sIL-6R), a process that has been termed trans-signaling. Recently, CD5 was described as an alternative alpha-receptor for IL-6 on B cells leading to the phosphorylation of the transcription factor STAT3 via the signal-transducing ß-receptor gp130 in a Jak2-dependent manner. In this study, we sought to investigate whether IL-6 was also able to signal via soluble CD5 (sCD5) analogous to IL-6 trans-signaling. We show that IL-6 indeed binds to sCD5, but that this does not lead to the activation of signal transduction or cell proliferation. Furthermore, sCD5 did also not interfere with IL-6 classic signaling, suggesting that the affinity between the two proteins was too weak to provoke a biological effect. Thus, trans-signaling of IL-6 can only occur via sIL-6R, but not sCD5.

Proteolytic Origin of the Soluble Human IL-6R In Vivo and a Decisive Role of N-Glycosylation.

Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.

Clinical significance of GCF sIL-6R and calprotectin to evaluate the periodontal inflammation.

Background Periodontitis is an inflammatory disease. The aim of this study was to investigate whether the soluble form of interleukin-6 receptor (sIL-6R) and calprotectin concentrations in gingival crevicular fluid are useful biomarkers in the evaluation of periodontitis. Methods First, a cross-sectional study was performed. A total of 34 periodontitis patients were enrolled and the gingival crevicular fluid samples were collected from the healthy and inflamed sites of periodontal pockets in each patient. The relationship between periodontal condition and gingival crevicular fluid sIL-6R and calprotectin concentrations was analysed statistically. The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontitis were determined using a receiver operating characteristic curve. Next, by using enzyme-linked immunosorbent assay, it was examined whether calprotectin induces sIL-6R production in THP-1 macrophages. Results Both gingival crevicular fluid sIL-6R and calprotectin concentrations were significantly higher in the inflamed sites than in the healthy sites ( P < 0.0001). The cut-off values of gingival crevicular fluid sIL-6R and calprotectin concentrations for the evaluation of periodontal inflammation were as follows: sIL-6R: 43.5 pg/site; calprotectin: 134.3 ng/site. In the in vitro study, calprotectin significantly induced sIL-6R production in THP-1 macrophages ( P < 0.01). Conclusions Both gingival crevicular fluid sIL-6R and calprotectin concentrations are significant biomarkers in the evaluation of periodontal inflammation.

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies.

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe(229) and Phe(279) of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe(279) Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe(279) In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe(279), whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe(229).

SELEX of Cell-Specific RNA Aptamers.

This chapter focuses on the selection of RNA aptamers, which bind to specific cell surface components and thus can be internalized receptor mediated. Such aptamers discriminate between different tissues, e.g., detect malignant cells, and target them or induce apoptosis through drug internalization. However, before starting the selection process the choice of an ideal target can be challenging. To give an example for the selection of cell specific aptamers, we here used the interleukin-6 receptor (IL-6R) as a target, which is presented on hepatocytes, neutrophils, monocytes, and macrophages.

SPECT imaging of interleukin-6 receptor in ovarian tumor xenografts with a novel radiotracer of 99mTc-HYNIC-Aca-LSLITRL.

Growing evidences have shown that the IL-6/IL-6R signal pathway promotes the tumor growth, angiogenesis, invasion and migration in various cancers, especially for epithelial ovarian cancer. Hence, including anti-IL-6 antibody (Siltuximab) and anti-IL-6R antibody (Tocilizumab), more and more therapeutic drugs targeting IL-6/IL-6R pathway were developed to block their activity. The molecular imaging of IL-6R is a significant factor for predicting tumor response to IL-6/IL-6R targeted drugs. However, few probes targeting IL-6R were designed and used for the specific detection. The purpose of this study was to develop and evaluate a novel radiotracer, (99m)Tc-HYNIC-Aca-LSLITRL, for SPECT imaging of interleukin-6 receptor. The expression of IL-6R was determined by western blot, immunofluorescence and immunohistochemistry. HYNIC-Aca-LSLITRL and HYNIC-Aca-TLQASIL were synthesized, and then were labeled with 99mTc. The stability and the cell-binding assay were performed. Ovarian tumor xenografts were established and subjected to SPECT imaging after injection of these two radiopharmaceuticals with or without excess primary peptides. The biodistribution of these two radiotracers was performed in nude mice bearing C13K tumors. (99m)Tc-HYNIC-Aca-LSLITRL and (99m)Tc-HYNIC-Aca-TLQASIL were obtained in >95 % labeling yield with favorably stability. In vitro studies demonstrated that the interleukin-6 receptor was overexpressed in ovarian cancer C13K cells. The SPECT imaging of interleukin-6 receptor and biodistribution studies showed that (99m)Tc-HYNIC-Aca-LSLITRL had higher tumor uptake and significantly lower kidney accumulation compared to (99m)Tc-HYNIC-Aca-TLQASIL. (99m)Tc-HYNIC-Aca-LSLITRL could be a promising agent for SPECT imaging of interleukin-6 receptor of ovarian cancer especially for those anti-IL-6R drugs under clinical trials, such as tocilizumab.

Calcium-dependent antigen binding as a novel modality for antibody recycling by endosomal antigen dissociation.

The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody.

Modular organization of Interleukin-6 and Interleukin-11 α-receptors.

Interleukin (IL)-6 and IL-11 are the only canonical members of the IL-6 family of cytokines that induce signaling through a homodimer of the common ß-receptor glycoprotein (gp)130. A pre-requisite for signal transduction is the initial binding of the cytokines to their unique α-receptors, IL-6R and IL-11R. The cell-type specific expression of the two receptors determines the target cells of IL-6 and IL-11, because gp130 is ubiquitously expressed. However, ciliary neurotrophic factor (CNTF) and IL-27p28/IL-30 have been described as additional ligands for the IL-6R, underlining a remarkable plasticity among the cytokines of the IL-6 family and their receptors. In this study, we show that neither IL-6 nor IL-11 can bind to and signal through the α-receptor of the respective other cytokine. We further create eight chimeric IL-6/IL-11 receptors, which are all biologically active. We find that the domains D1 to D3, which contain the cytokine binding module (CBM), determine which cytokine can activate the chimeric receptor, whereas the stalk region, the transmembrane region, or the intracellular region do not participate in the ligand selectivity of the receptor and are therefore interchangeable between IL-6R and IL-11R. These results suggest a modular organization of the IL-6R and IL-11R, and a similar signal transduction complex of the two cytokines.