The Janus kinase/STAT 1 pathway plays a major role in cytokine and growth factor signaling. In particular, the family members of the ␣-helix bundle cytokines comprising the hematopoietins and interferons exert their biological effects by the activation of STAT transcription factors (for review, see Ref. 1). According to the canonical model (2), signaling through the Janus kinase/STAT pathway is triggered by the binding of a cytokine to class I or class II cytokine receptors which leads to the activation of receptor-associated tyrosine kinases of the Janus kinase family. The activated Janus kinases phosphorylate the receptor on tyrosine residues followed by the recruitment of STAT monomers to these phosphorylated tyrosine motifs. While bound to the receptor, STATs are phosphorylated at a single tyrosine residue and subsequently form dimers by intermolecular phosphotyrosine-SH2 domain interactions. The STAT dimers freely diffuse through the cytosol (3), associate with importin-␣ (4), and translocate via the nuclear pore complexes to the nuclear compartment to transactivate target genes.This canonical model has been challenged by accumulating data suggesting that STATs dimerize prior to activation. The preformation of STAT dimers seems to be independent of tyrosine phosphorylation (5-10). Furthermore, recent observations from several laboratories suggest that a subpopulation of STAT proteins is located in the nucleus of unstimulated cells (8, 10 -15). The latter finding is either the result of a static subcellular distribution or a continuous dynamic shuttling of STAT molecules between the cytosol and the nucleus.STAT3 is one of the seven mammalian STAT proteins. It acts as a signal transducer for many cytokines and growth factors (16) and is of particular importance for the family of IL-6-type cytokines (17). STAT3 participates in a variety of biological processes such as the induction of acute phase protein synthesis in hepatocytes (18), the regulation of hematopoiesis and the immune response (16,19), embryo implantation (20, 21), and development (22, 23). As a consequence of these diverse functions, STAT3 plays a crucial role in inflammatory, autoimmune, and certain neoplastic diseases (24). Mice having a targeted deletion of STAT3 die early during embryonic development (22).STAT proteins consist of six domains: an N-terminal domain involved in cooperative DNA binding, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a C-terminal transactivation domain. The structure of a truncated, tyrosine-phosphorylated, dimeric STAT3 bound to DNA has been solved by x-ray crystallography (25). The dimerization of STAT3 molecules is enforced by reciprocal binding of the phosphotyrosine-containing regions to the SH2 domains, which is a prerequisite for nuclear accumulation and DNA binding of STAT3. The activity of the C-terminal transactivation domain is modulated by phosphorylation at serine 727 (26).The major part of the data concerning the nucleocytoplasmic transport mechanisms of STAT protein...

Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 2∶1 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.dsFv | imaging | immunotoxin | protein engineering

The cytokine receptor gp130 is the shared signalling subunit of the IL-6-type cytokines. Interleukin-6 (IL-6) signals through gp130 homodimers whereas leukaemia inhibitory factor (LIF) exerts its action through a heterodimer of gp130 and the LIF receptor (LIFR). Related haematopoietic receptors such as the erythropoietin receptor have been described as preformed dimers in the plasma membrane. Here we investigated gp130 homodimerization and heterodimerization with the LIFR by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC). We detected a FRET signal between YFP- and CFP-tagged gp130 at the plasma membrane of unstimulated cells that does not increase upon IL-6 stimulation. However, FRET between YFP-tagged gp130 and CFP-tagged LIFR considerably increased upon LIF stimulation. Using a BiFC approach that detects stable interactions we show that fluorescence complementation of gp130 constructs tagged with matching `halves' of fluorescent proteins increases upon IL-6 stimulation. Taken together, these findings suggest that transient gp130 homodimers on the plasma membrane are stabilized by IL-6 whereas heterodimerization of gp130 with the LIFR is mainly triggered by the ligand. This view is supported by the observation that the simultaneous action of two IL-6 binding domains on two gp130 molecules is required to efficiently recruit a fluorescent IL-6 (YFP-IL-6) to the plasma membrane.

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one VH and one VL domain connected by a disulfide bond. The molecules are assembled by fusing a VH,Cys44 domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a VL,Cys100 to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between VH and VL. Tethering the VH and VL domains at the C-terminus of the CH3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.

Proinflammatory cytokines such as tumor necrosis factor (TNF), 2 interleukin-1␤ (IL-1␤), or interleukin-6 (IL-6) have been identified as promising therapeutic targets in the treatment of chronic inflammation. A dimeric soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1). Whereas TNF signals through a receptor homotrimer, most cytokines signal through receptor complexes consisting of two or more different receptor subunits. In this case, the respective cytokine can be inhibited by using fusion proteins composed of the different soluble receptors, as we and others showed for the inhibition of IL-6 (2-4).All cytokines signaling through the common receptor subunit gp130 belong to the family of IL-6-type cytokines (5), which includes IL-6, IL-11, IL-27, LIF, OSM, ciliary neurotrophic factor, cardiotrophin-1, cardiotophin-like cytokine, and neuropoietin. IL-6-type cytokines contain distinct receptorbinding sites that were discovered by mutagenesis studies on IL-6, ciliary neurotrophic factor, and LIF (6 -8). The IL-6-type cytokines can be subdivided into those containing three (I, II, and III) or two (II and III) receptor-binding sites. Site I determines the specificity of ␣-receptor binding. The ␣-receptor is not capable of transferring the signal into the cell but is crucial for increasing the binding affinity of the cytokine to its signaling receptors. Site II seems to be the universal gp130-binding site of all IL-6-type cytokines. Depending on the cytokine, site III is used for the recruitment of LIFR, OSMR, or a second gp130 molecule (5). The IL-6 inhibitor IL-6-RFP (2, 3) was designed to block a cytokine containing all three receptor-binding sites.However, there are also IL-6-type cytokines, which do not need to recruit an ␣-receptor analogous to human IL-6R␣, and thus do not seem to have a functional site I. One example for a cytokine belonging to this group is the leukemia inhibitory factor (LIF). In this study we present an approach to construct inhibitory receptor fusion proteins for human and murine LIF as prototypes of inhibitors targeting cytokines whose receptors only bind to the site II and III of the cytokine without occupying site I. We designate these inhibitors "site II/III inhibitors."LIF signals through a heterodimer of LIFR and gp130. Janus tyrosine kinases that are constitutively associated with the cytoplasmic parts of gp130 (9) and LIFR (10) are activated upon ligand binding and phosphorylate the receptors and the recruited transcription factor STAT3. Activated STAT3 dimerizes and translocates into the nucleus, where it induces LIF target genes (11).We wanted to integrate only those receptor domains of gp130 and LIFR into the inhibitory receptor fusion proteins that are necessary for LIF binding. For gp130, which includes six extracellular domains (D1-D6), it has been clearly shown that domains D2 and D3 forming the cytokine-binding module (CBM) are necessary and sufficient for LIF binding (12). In contrast, there are contradi...

Although fusion proteins of the extracellular parts of receptor subunits termed cytokine traps turned out to be promising cytokine inhibitors for anti-cytokine therapies, their mode of action has not been analyzed. We developed a fusion protein consisting of the ligand binding domains of the IL-6 receptor subunits IL-6R␣ and gp130 that acts as a highly potent IL-6 inhibitor. Gp130 is a shared cytokine receptor also used by the IL-6-related cytokines oncostatin M and leukemia inhibitory factor. In this study, we have shown that the IL-6 receptor fusion protein (IL-6-RFP) is a specific IL-6 inhibitor that does not block oncostatin M or leukemia inhibitory factor. We characterized the complex of IL-6-RFP and fluorescently labeled IL-6 (YFP-IL-6) by blue native PAGE and gel filtration. A 2-fold molar excess of IL-6-RFP over IL-6 was sufficient to entirely bind IL-6 in a complex with IL-6-RFP. As shown by treatment with urea and binding competition experiments, the complex of IL-6 and IL-6-RFP is more stable than the complex of IL-6, soluble IL-6R␣, and soluble gp130. By live cell imaging, we have demonstrated that YFP-IL-6 bound to the surface of cells expressing gp130-CFP is removed from the plasma membrane upon the addition of IL-6-RFP. The apparent molecular mass of the IL-6⅐IL-6-RFP complex determined by blue native PAGE and gel filtration suggests that IL-6 is trapped in a structure analogous to the native hexameric IL-6 receptor complex. Thus, fusion of the ligand binding domains of heteromeric receptors leads to highly specific cytokine inhibitors with superior activity compared with the separate soluble receptors.Cytokines are important mediators in the regulation of immune responses and inflammation. Dysregulated cytokine signaling leads to chronic inflammation and cancer. Therefore, pro-inflammatory cytokines, such as tumor necrosis factor (TNF) 2 and interleukin-1 and -6, have been identified as promising therapeutic targets. First approaches to specifically block the action of pro-inflammatory cytokines have focused on the use of neutralizing antibodies against a specific cytokine or its receptor. Only recently, the value of soluble cytokine receptors as cytokine antagonists for the treatment of inflammatory diseases has been fully recognized. Pro-inflammatory cytokines signal through receptor proteins consisting of an extracellular part, a single transmembrane region and a cytoplasmic domain. Ligand binding to the extracellular part of the receptor results in the activation of signal transduction cascades by the cytoplasmic domain. Soluble receptors consisting only of the extracellular part are potent inhibitors of cytokine activity. They bind the cytokine with the same specificity and affinity as the membranebound receptors without eliciting an intracellular signal. A dimeric form of the soluble TNF receptor is currently used for the treatment of inflammatory diseases caused by elevated TNF expression (1).Most cytokines signal through heteromeric receptor complexes consisting of two or more different r...

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