Acid-sensing ion channels (ASICs) have emerged as important, albeit challenging therapeutic targets for pain, stroke, etc. One approach to developing therapeutic agents could involve the generation of functional antibodies against these channels. To select such antibodies, we used channels assembled in nanodiscs, such that the target ASIC1a has a configuration as close as possible to its natural state in the plasma membrane. This methodology allowed selection of functional antibodies that inhibit acid-induced opening of the channel in a dose-dependent way. In addition to regulation of pH, these antibodies block the transport of cations, including calcium, thereby preventing acid-induced cell death in vitro and in vivo. As proof of concept for the use of these antibodies to modulate ion channels in vivo, we showed that they potently protect brain cells from death after an ischemic stroke. Thus, the methodology described here should be general, thereby allowing selection of antibodies to other important ASICs, such as those involved in pain, neurodegeneration, and other conditions.

Cytokines are protein mediators that are known to be involved in many biological processes, including cell growth, survival, inflammation, and development. To study their regulation, we generated a library of 209 different cytokines. This was used in a combinatorial format to study the effects of cytokines on each other, with particular reference to the control of differentiation. This study showed that IFN-γ is a master checkpoint regulator for many cytokines. It operates via an autocrine mechanism to elevate STAT1 and induce internalization of gp130, a common component of many heterodimeric cytokine receptors. This targeting of a receptor subunit that is common to all members of an otherwise diverse family solves the problem of how a master regulator can control so many diverse receptors. When one adds an autocrine mechanism, fine control at the level of individual cells is achieved.A s our understanding of physiology grows, we are increasingly aware of its Newtonian aspects in that for every action there is another reaction with opposite consequences (1). Irrespective of whether one thinks in terms of homeostasis, feedback loops, or checkpoints, we increasingly uncover molecular systems that, in terms of function, induce cells to move in opposite ways. For example, although immune T-cell activation is critical in controlling disease, one needs a checkpoint to guard against overactivity that could result in autoimmunity (2, 3).In addition to adding to our general knowledge of cellular physiology, detailed understanding of the molecular mechanisms of activation and checkpoint processes has important therapeutic implications. Such an understanding allows two separate entry points into the regulation of cellular events. Thus, if one wants to promote a cellular function, the same outcome can be achieved by either enhancing the effector or inhibiting the checkpoint pathways, usually by perturbing the molecules that initiate them.One of the most important physiological systems is the cytokine cascade (4). However, here, because the cascade contains large numbers of separate molecules, each operating through different receptors, simple models of regulation break down. While one can imagine that each cytokine is paired with a separate checkpoint system operating in an opposite direction, it seemed to us to be unlikely, if for no other reason than the economical use of genetic information. Given that one had large sets of related molecules with overlapping signal transduction mechanisms and pathways, it seemed more reasonable to assume that, depending on the circumstance, some members of the cytokine repertoire regulate other members of the repertoire. This differs from situations such as PD-1, where the checkpoint mechanistic cascade differs completely from the activation mechanism. By contrast, we propose that cytokine members of a family regulate each other by perturbing common molecular mechanisms. Thus, if gene action economy is proposed for a family, its hallmark should be that some of the same molecules used for...

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Tspan12/β-catenin signaling is critical for the progression of vasoproliferative disease. The newly developed anti-Tspan12 antibody has therapeutic effects in vasoproliferative retinopathy and can enhance the potency of existing anti- vascular endothelial growth factor agents.

p185her2/neu belongs to the ErbB receptor tyrosine kinase family, which has been associated with human breast, ovarian, and lung cancers. Targeted therapies employing ectodomain-specific p185 her2/neu monoclonal antibodies (mAbs) have demonstrated clinical efficacy for breast cancer. Our previous studies have shown that p185 her2/neu mAbs are able to disable the kinase activity of homomeric and heteromeric kinase complexes and induce the conversion of the malignant to normal phenotype. We previously developed a chimeric antibody chA21 that specifically inhibits the growth of p185 her2/neu -overexpressing cancer cells in vitro and in vivo. Herein, we report the crystal structure of the single-chain Fv of chA21 in complex with an N-terminal fragment of p185 her2/neu , which reveals that chA21 binds a region opposite to the dimerization interface, indicating that chA21 does not directly disrupt the dimerization. In contrast, the bivalent chA21 leads to internalization and down-regulation of p185 her2/neu . We propose a structure-based model in which chA21 cross-links two p185 her2/neu molecules on separate homo-or heterodimers to form a large oligomer in the cell membrane. This model reveals a mechanism for mAbs to drive the receptors into the internalization/degradation path from the inactive hypophosphorylated tetramers formed dynamically by active dimers during a "physiologic process." p185 her2/neu is one of the four receptor tyrosine kinases of the ErbB family. We initially found that both homodimerization and heterodimerization with other ErbB receptors will induce transphosphorylation of the intracellular domains and result in the downstream signaling for cell proliferation and transformation. Moreover our studies have established that heterodimerization leads to increased signaling and transforming activity (1, 2). Significant overexpression of p185 her2/neu results in abnormalities in cell signaling and can cause cell transformation. Early studies from several laboratories found that her2/neu gene was amplified and overexpressed in 20 -30% of breast and ovarian cancers. Breast cancers that have p185 her2/neu overexpressed have a more aggressive course associated with higher relapse rates (3). p185her2/neu represents the first oncoprotein target amenable for drug intervention and immunotherapy in which disabling the kinase reverses the malignant properties of the transformed cell and renders the tumor sensitive to chemotherapy and radiation therapy (4 -8).The p185 her2/neu protein possesses a similar architecture to the other three ErbB members of this family. These kinases are type 1 transmembrane proteins and comprise an extracellular domain (ECD) 4 with four subdomains (I/L1, II/S1, III/L2, IV/S2), a single transmembrane helix, an intracellular tyrosine kinase domain, and a C-terminal tail (9). Recent crystallographic studies revealed that the subdomains II and IV contribute to dimerization events of the ErbB receptors (10, 11). Monoclonal antibodies that bind the ectodomains of these ErbB proteins hav...

Growth factor deficiency in adulthood constitutes a distinct clinical syndrome with significant morbidities including abnormal body composition, reduced energy, affective disturbances, dyslipidemia, and increased cardiovascular risk. Protein replacement therapies using recombinant proteins or enzymes represent the only approved treatment. Combinatorial antibodies have shown great promise as a new class of therapeutic molecules because they act as “mechanism‐based antibodies” with both agonist and antagonist activities. Using leptin, a key hormone in energy metabolism, as an example, a function‐guided approach is developed to select combinatorial antibodies with high potency and full agonist activity that substitute natural growth factors in vivo. The identified antibody shows identical biochemical properties and cellular profiles as leptin, and rescues leptin‐deficiency in ob/ob mice. Remarkably, the antibody activates leptin receptors that are otherwise nonfunctional because of mutations (L372A and A409E). Combinatorial antibodies have significant advantages over recombinant proteins for chronical usage in terms of immunological tolerance and biological stability.

Background Rhinoviruses (RVs) cause more than half of common colds and, in some cases, more severe diseases. Functional genomics analyses of RVs using siRNA or genome-wide CRISPR screen uncovered a limited set of host factors, few of which have proven clinical relevance. Results Herein, we systematically compare genome-wide CRISPR screen and surface protein-focused CRISPR screen, referred to as surfaceome CRISPR screen, for their efficiencies in identifying RV host factors. We find that surfaceome screen outperforms the genome-wide screen in the success rate of hit identification. Importantly, using the surfaceome screen, we identify olfactomedin-like 3 (OLFML3) as a novel host factor of RV serotypes A and B, including a clinical isolate. We find that OLFML3 is a RV-inducible suppressor of the innate immune response and that OLFML3 antagonizes type I interferon (IFN) signaling in a SOCS3-dependent manner. Conclusion Our study suggests that RV-induced OLFML3 expression is an important mechanism for RV to hijack the immune system and underscores surfaceome CRISPR screen in identifying viral host factors.

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