Inhibition of GSK-3 ameliorates the pathogenesis of Huntington's disease.

In Huntington's disease (HD), the mutant huntingtin (mHtt) accumulates as toxic aggregates in the striatum tissue, with deleterious effects on motor-coordination and cognitive functions. Reducing the levels of mHtt is therefore a promising therapeutic strategy. We have previously reported that GSK-3 is a negative regulator of the autophagy/lysosome pathway, which is responsible for intracellular degradation, and is critically important for maintaining neuronal vitality. Thus, we hypothesized that inhibition of GSK-3 may trigger mHtt clearance thereby reducing mHtt cytotoxicity and improving HD symptoms. Here, we demonstrate that depletion or suppression of autophagy results in a massive accumulation of mHtt aggregates. Accordingly, mHtt aggregates were localized in lysosomes, but, mostly mislocalized from lysosomes in the absence of functional autophagy. Overexpression of GSK-3, particularly the α isozyme, increased the number of mHtt aggregates, while silencing GSK-3α/ß, or treatment with a selective GSK-3 inhibitor, L807mts, previously described by us, reduced the amounts of mHtt aggregates. This effect was mediated by increased autophagic and lysosomal activity. Treating R6/2 mouse model of HD with L807mts, reduced striatal mHtt aggregates and elevated autophagic and lysosomal markers. The L807mts treatment also reduced hyperglycemia and improved motor-coordination functions in these mice. In addition, L807mts restored the expression levels of Sirt1, a critical neuroprotective factor in the HD striatum, along with its targets BDNF, DRPP-32, and active Akt, all provide neurotrophic/pro-survival support and typically decline in the HD brain. Our results provide strong evidence for a role for GSK-3 in the regulation of mHtt dynamics, and demonstrate the benefits of GSK-3 inhibition in reducing mHtt toxicity, providing neuroprotective support, and improving HD symptoms.

"BIClonals": Production of Bispecific Antibodies in IgG Format in Transiently Transfected Mammalian Cells.

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components. For some therapeutic applications, full-size, native IgG-like bsAbs may be the optimal format.To prepare bsAbs in IgG format, two challenges should be met. One is that each heavy chain will only pair with the heavy chain of the second specificity and that heavy chain homodimerization will be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and that pairing with the light chain of the second specificity will be prevented. The first solution to the first criterion (known as knobs into holes, KIH) was presented in 1996 by Genentech and additional solutions were presented more recently. However, until recently, out of >120 published formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested.Here, we present a protocol for preparing bsAbs in IgG format in transfected mammalian cells. For heavy chain dimerization we use KIH while as a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs we present our "BIClonals" technology; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL.During our studies of bsAbs we found that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies; hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs that suits every antibody sequence, making it necessary to carefully evaluate the optimal solution for each new antibody.

Production of Stabilized Antibody Fragments in the E. coli Bacterial Cytoplasm and in Transiently Transfected Mammalian Cells.

Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the periplasm bacterium E. coli. The production of soluble scFvs is more effective in quantity, however, under the reducing conditions of the E. coli bacterial cytoplasm it is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMALc-TNN and pMALc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by Tobacco Etch Virus (TEV) protease in order to obtain untagged scFv.The second protocol is for efficient production of Fab antibody fragments as MBP fusion proteins secreted by transiently transfected mammalian cells. While transient transfection is a fast and effective way of obtaining several mgs of antibody for initial screening and validation of antibodies, some antibody sequences express poorly or not at all. For such antibodies, fusion to MBP provides an effective approach for solving the expression problem.

Design Principles for Bispecific IgGs, Opportunities and Pitfalls of Artificial Disulfide Bonds.

Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not achievable with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated, but as many of them are small recombinant fragments, their utility could be limited. For some therapeutic applications, full-size IgGs may be the optimal format. Two challenges should be met to make bispecific IgGs; one is that each heavy chain will only pair with the heavy chain of the second specificity and that homodimerization be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and not with the light chain of the second specificity. The first solution to the first criterion (knobs into holes, KIH) was presented in 1996 by Paul Carter's group from Genentech. Additional solutions were presented later on. However, until recently, out of >120 published bsAb formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested. We present a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL. We name antibodies produced according to this design "BIClonals". Bispecific IgGs where the artificial disulfide bond is placed in the CH1-CL interface are also presented. Briefly, we found that an artificial disulfide bond between VH position 44 to VL position 100 provides for effective and correct H-L chain pairing while also preventing the formation of wrong H-L chain pairs. When the artificial disulfide bond links the CH1 with the CL domain, effective H-L chain pairing also occurs, but in some cases, wrong H-L pairing is not totally prevented. We conclude that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies, hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs, making it necessary to carefully evaluate the optimal solution for each new antibody.

A unique type of GSK-3 inhibitor brings new opportunities to the clinic.

Development of protein kinase inhibitors is a focus of many drug discovery programs. A major problem, however, is the limited specificity of the commonly used adenosine triphosphate-competitive inhibitors and the weak inhibition of the more selective substrate-competitive inhibitors. Glycogen synthase kinase-3 (GSK-3) is a promising drug target for treating neurodegenerative disorders, including Alzheimer's disease (AD), but most GSK-3 inhibitors have not reached the clinic. We describe a new type of GSK-3 inhibitor, L807mts, that acts through a substrate-to-inhibitor conversion mechanism that occurs within the catalytic site of the enzyme. We determined that L807mts was a potent and highly selective GSK-3 inhibitor with reasonable pharmacological and safety properties when tested in rodents. Treatment with L807mts enhanced the clearance of ß-amyloid loads, reduced inflammation, enhanced autophagic flux, and improved cognitive and social skills in the 5XFAD AD mouse model. This new modality of GSK-3 inhibition may be therapeutic in patients with AD or other central nervous system disorders associated with dysregulated GSK-3.

Production of stabilized scFv antibody fragments in the E. coli bacterial cytoplasm.

Monoclonal antibodies (mAbs) are currently the fastest growing class of therapeutic proteins. Parallel to full-length IgG format the development of recombinant technologies provided the production of smaller recombinant antibody variants. The single-chain variable fragment (scFv) antibody is a minimal form of functional antibody comprised of the variable domains of immunoglobulin light and heavy chains connected by a flexible linker. In most cases, scFvs are expressed in the bacterium E. coli. The production of soluble scFvs under the reducing conditions of the E. coli bacterial cytoplasm is inefficient because of the inability of the disulfide bonds to form. Hence, scFvs are either secreted to the periplasm as soluble proteins or expressed in the cytoplasm as insoluble inclusion bodies and recovered by refolding. The cytoplasmic expression of scFvs as a C-terminal fusion to maltose-binding protein (MBP) provided the high-level production of stable, soluble, and functional fusion protein. The below protocol provides the detailed description of MBP-scFv production in E. coli utilizing two expression systems: pMalc-TNN and pMalc-NHNN. Although the MBP tag does not disrupt the most of antibody activities, the MBP-TNN-scFv product can be cleaved by TEV protease in order to obtain untagged scFv.

Phenylalanine assembly into toxic fibrils suggests amyloid etiology in phenylketonuria.

Phenylketonuria (PKU) is characterized by phenylalanine accumulation and progressive mental retardation caused by an unknown mechanism. We demonstrate that at pathological concentrations, phenylalanine self-assembles into fibrils with amyloid-like morphology and well-ordered electron diffraction. These assemblies are specifically recognized by antibodies, show cytotoxicity that can be neutralized by the antibodies and are present in the hippocampus of model mice and in parietal cortex brain tissue from individuals with PKU. This is, to our knowledge, the first demonstration that a single amino acid can form amyloid-like deposits, suggesting a new amyloidosis-like etiology for PKU.

In vivo characteristics of targeted drug-carrying filamentous bacteriophage nanomedicines.

BACKGROUND: Targeted drug-carrying phage nanomedicines are a new class of nanomedicines that combines biological and chemical components into a modular nanometric drug delivery system. The core of the system is a filamentous phage particle that is produced in the bacterial host Escherichia coli. Target specificity is provided by a targeting moiety, usually an antibody that is displayed on the tip of the phage particle. A large drug payload is chemically conjugated to the protein coat of the phage via a chemically or genetically engineered linker that provides for controlled release of the drug after the particle homed to the target cell. Recently we have shown that targeted drug-carrying phage nanomedicines can be used to eradicate pathogenic bacteria and cultured tumor cells with great potentiation over the activity of the free untargeted drug. We have also shown that poorly water soluble drugs can be efficiently conjugated to the phage coat by applying hydrophilic aminoglycosides as branched solubility-enhancing linkers. RESULTS: With an intention to move to animal experimentation of efficacy, we tested anti-bacterial drug-carrying phage nanomedicines for toxicity and immunogenicity and blood pharmacokinetics upon injection into mice. Here we show that anti-bacterial drug-carrying phage nanomedicines that carry the antibiotic chloramphenicol conjugated via an aminoglycoside linker are non-toxic to mice and are greatly reduced in immunogenicity in comparison to native phage particles or particles to which the drug is conjugated directly and are cleared from the blood more slowly in comparison to native phage particles. CONCLUSION: Our results suggest that aminoglycosides may serve as branched solubility enhancing linkers for drug conjugation that also provide for a better safety profile of the targeted nanomedicine.

Antibacterial application of engineered bacteriophage nanomedicines: antibody-targeted, chloramphenicol prodrug loaded bacteriophages for inhibiting the growth of Staphylococcus aureus bacteria.

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus there is an urgent need to develop new antimicrobial agents. To be effective, these new antimicrobials should possess novel modes of action and/or different cellular targets compared with existing antibiotics. Bacteriophages (phages) have been used for over a century as tools for the treatment of bacterial infections, for nearly half a century as tools in genetic research, for about two decades as tools for the discovery of specific target-binding proteins and peptides, and for almost a decade as tools for vaccine development. We describe a new application in the area of antibacterial nanomedicines where filamentous phages can be formulated as targeted drug-delivery vehicles of nanometric dimensions (phage nanomedicines) and used for therapeutic purposes. This protocol involves both genetic and chemical engineering of these phages. The genetic engineering of the phage coat, which results in the display of a target-specificity-conferring peptide or protein on the phage coat, can be used to design the drug-release mechanism and is not described herein. However, the methods used to chemically conjugate cytotoxic drugs at high density on the phage coat are described. Further, assays to measure the drug load on the surface of the phage and the potency of the system in the inhibition of growth of target cells as well as assessment of the therapeutic potential of the phages in a mouse disease model are discussed.