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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.