Group A streptococcal (GAS) infections and autoimmunity are associated with the onset of a spectrum of neuropsychiatric disorders in children, with the prototypical disorder being Sydenham chorea (SC). Our aim was to develop an animal model that resembled the behavioral, pharmacological, and immunological abnormalities of SC and other streptococcal-related neuropsychiatric disorders. Male Lewis rats exposed to GAS antigen exhibited motor symptoms (impaired food manipulation and beam walking) and compulsive behavior (increased induced-grooming). These symptoms were alleviated by the D2 blocker haloperidol and the selective serotonin reuptake inhibitor paroxetine, respectively, drugs that are used to treat motor symptoms and compulsions in streptococcal-related neuropsychiatric disorders. Streptococcal exposure resulted in antibody deposition in the striatum, thalamus, and frontal cortex, and concomitant alterations in dopamine and glutamate levels in cortex and basal ganglia, consistent with the known pathophysiology of SC and related neuropsychiatric disorders. Autoantibodies (IgG) of GAS rats reacted with tubulin and caused elevated calcium/calmodulindependent protein kinase II signaling in SK-N-SH neuronal cells, as previously found with sera from SC and related neuropsychiatric disorders. Our new animal model translates directly to human disease and led us to discover autoantibodies targeted against dopamine D1 and D2 receptors in the rat model as well as in SC and other streptococcal-related neuropsychiatric disorders.

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Previous studies have identified relevant genes and signalling pathways that are hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as short interfering RNAs (siRNAs), opens up opportunities for personalized medicine. Yet, although major progress has been made in developing siRNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting, their clinical translation has not occurred. This is in part because of the massive development and production requirements and the high batch-to-batch variability of current technologies, which rely on chemical conjugation. Here we present a self-assembled modular platform that enables the construction of a theoretically unlimited repertoire of siRNA targeted carriers. The self-assembly of the platform is based on a membrane-anchored lipoprotein that is incorporated into siRNA-loaded lipid nanoparticles that interact with the antibody crystallizable fragment (Fc) domain. We show that a simple switch of eight different mAbs redirects the specific uptake of siRNAs by diverse leukocyte subsets in vivo. The therapeutic potential of the platform is demonstrated in an inflammatory bowel disease model by targeting colon macrophages to reduce inflammatory symptoms, and in a Mantle Cell Lymphoma xenograft model by targeting cancer cells to induce cell death and improve survival. This modular delivery platform represents a milestone in the development of precision medicine.

While the resistance of bacteria to traditional antibiotics is a major public health concern, the use of extremely potent antibacterial agents is limited by their lack of selectivity. As in cancer therapy, antibacterial targeted therapy could provide an opportunity to reintroduce toxic substances to the antibacterial arsenal. A desirable targeted antibacterial agent should combine binding specificity, a large drug payload per binding event, and a programmed drug release mechanism. Recently, we presented a novel application of filamentous bacteriophages as targeted drug carriers that could partially inhibit the growth of Staphylococcus aureus bacteria. This partial success was due to limitations of drug-loading capacity that resulted from the hydrophobicity of the drug. Here we present a novel drug conjugation chemistry which is based on connecting hydrophobic drugs to the phage via aminoglycoside antibiotics that serve as solubility-enhancing branched linkers. This new formulation allowed a significantly larger drugcarrying capacity of the phages, resulting in a drastic improvement in their performance as targeted drug-carrying nanoparticles. As an example for a potential systemic use for potent agents that are limited for topical use, we present antibody-targeted phage nanoparticles that carry a large payload of the hemolytic antibiotic chloramphenicol connected through the aminoglycoside neomycin. We demonstrate complete growth inhibition toward the pathogens Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli with an improvement in potency by a factor of ϳ20,000 compared to the free drug.The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels (2, 3), spurring a strong need to develop new antimicrobial agents. Classical short-term approaches include chemical modification of existing agents to improve potency or spectrum. Long-term approaches rely on bacterial and phage genomics to discover new antibiotics that attack new protein targets which are essential to bacterial survival and therefore with no known resistance (1, 8). In both traditional and newly developed antibiotics, the target selectivity lies in the drug itself, in its ability to affect a mechanism that is unique to the target microorganism and absent in its host. As a result, a vast number of potent drugs have been excluded from use as therapeutics due to low selectivity. This brings to mind the limited selectivity of anticancer drugs and recent efforts to overcome it by developing targeted therapeutic strategies. Antibody-based targeted drug delivery approaches have been developed since the advent of monoclonal antibodies (6). Since then, monoclonal antibodies and derived single-chain antibodies were used to deliver potent cytotoxic components to cancer cells that, once bound, internalize and kill the target cell (7, 12). A similar immunotargeting of bacteria is not feasible due to the lack of a bacterial internalization process, making the use of an extracellular release mechanism ...

The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound Pseudomonas exotoxin A (PE) belongs to a family of bacterial nicotinamide adenine dinucleotide (NAD)-binding toxins that ADP ribosylate target proteins within eukaryotic cells (1).Crystal structures have been reported for four of these toxins: PE (2), diphtheria toxin (DT) (3), Escherichia coli heat-labile enterotoxin (LT) (4), and pertussis toxin (PT) (5), revealing a common fold for the catalytic ADP ribosylating domain, consisting of two approximately orthogonal, antiparallel (3 sheets flanked by several helices forming a large substrate binding cleft at the center of the domain. The folding topology is unlike the dinucleotide binding fold observed in several dehydrogenases (6).PE is a 613-amino acids protein that is composed of three structural domains. Domain 1 (1-252 and 365-399) binds to the ubiquitous a2-macroglobulin receptor of eukaryotic cells and initiates receptor-mediated endocytosis (7). In the cell, PE is cleaved at residue 280 within domain II (residues 253 to 364) by a specific protease (8); this cleavage activates the third domain. The portion of domain II (residues 280-364) remaining with the C-terminal fragment appears to translocate the fragment through intracellular membranes into the cytosol, where domain III (residues 400-613) acts by transferring the ADP ribose from NAD to a modified histidine in elongation factor-2 (EF-2). This irreversible covalent modification inhibits protein synthesis and leads to cell death. The function of domain lb (residues 365-399) is unknown and it can be entirely deleted without loss of toxin activity (9).In vitro, the intact toxin molecule has low affinity for NAD and exhibits no ADP ribosyl transferase activity (2). Treatment with reducing and denaturing agents is necessary to obtain an active molecule that can both bind to NAD and effect ADP ribosylation of EF-2 (10). The isolated domain III (PEIII) is active in the absence of this activation treatment (11). PEIII also exhibits a weak NAD glycohydrolase activity (12). We have determined (13) the structure of this catalytic domain complexed with the products of NAD hydrolysis, AMP and nicotinamide, thus effectively defining the active site. This structure revealed that the process of activation must involve the removal of helix 333 to 353 of domain TI from the vicinity of the active site loop 458 to 463 of domain ITT, thus permitting a conformational change in this loop that is necessary for NAD binding.In this structure, although the crystals were grown in the presence of NAD, resulting hydrolysis prevented the v...

Recombinant single-chain antibodies (scFvs) that are expressed in the cytoplasm of cells are of considerable biotechnological and therapeutic potential. However, the reducing environment of the cytoplasm inhibits the formation of the intradomain disul®de bonds that are essential for correct folding and functionality of these antibody fragments. Thus, scFvs expressed in the cytoplasm are mostly insoluble and inactive.Here, we describe a general approach for stabilizing scFvs for ef®cient functional expression in the cell cytoplasm in a soluble, active form. The scFvs are expressed as C-terminal fusions with the Escherichia coli maltose-binding protein (MBP). We tested a large panel of scFvs that were derived from hybridomas and from murine and human scFv phage display and expression libraries by comparing their stability and functionality as un-fused versus MBP fused proteins. We found that MBP fused scFvs are expressed at high levels in the cytoplasm of E. coli as soluble and active proteins regardless of the redox state of the bacterial cytoplasm. In contrast, most un-fused scFvs can be produced (to much lower levels) in a functional form only when expressed in trxB À but not in trxB E. coli cells. We show that MBP-scFv fusions are more stable than the corresponding un-fused scFvs, and that they perform more ef®ciently in vivo as cytoplasmic intrabodies in E. coli. Thus, MBP seems to function as a molecular chaperone that promotes the solubility and stability of scFvs that are fused to it.

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Bacteriophages have been used for more than a century for (unconventional) therapy of bacterial infections, for half a century as tools in genetic research, for 2 decades as tools for discovery of specific target-binding proteins, and for nearly a decade as tools for vaccination or as gene delivery vehicles. Here we present a novel application of filamentous bacteriophages (phages) as targeted drug carriers for the eradication of (pathogenic) bacteria. The phages are genetically modified to display a targeting moiety on their surface and are used to deliver a large payload of a cytotoxic drug to the target bacteria. The drug is linked to the phages by means of chemical conjugation through a labile linker subject to controlled release. In the conjugated state, the drug is in fact a prodrug devoid of cytotoxic activity and is activated following its dissociation from the phage at the target site in a temporally and spatially controlled manner. Our model target was Staphylococcus aureus, and the model drug was the antibiotic chloramphenicol. We demonstrated the potential of using filamentous phages as universal drug carriers for targetable cells involved in disease. Our approach replaces the selectivity of the drug itself with target selectivity borne by the targeting moiety, which may allow the reintroduction of nonspecific drugs that have thus far been excluded from antibacterial use (because of toxicity or low selectivity). Reintroduction of such drugs into the arsenal of useful tools may help to combat emerging bacterial antibiotic resistance.