Limor Nahary

Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

An investigation of antistreptococcal antibody responses in guttate psoriasis

In two-thirds of patients with guttate psoriasis (GP), there is good evidence that the eruption is triggered by a streptococcal throat infection. We attempted to determine if a specific epitope of the bacterial pathogen was associated with the humoral immune response in GP patients. Antibody titres against beta-haemolytic streptococci (BHS) extracts in sera from 14 patients with GP, 10 healthy controls and 10 chronic plaque psoriasis (CPP) patients were determined by ELISA. Antibody BHS reactivity was investigated using immunoblotting, followed by epitope mapping using peptide-phage display. The highest GP antibody titres (10,000–25,000) were found in sera that had a matching streptococcal isolate, three sera had high (5,000–12,500) and seven had raised titres (500–5,000). In the healthy control group, three had relatively high and seven lower titres. All the CPP sera had very low titres (<500). In the immunoblots, three major bands were recognised by all the GP sera, and, to a lesser extent, by four healthy controls. No GP-specific protein was identified. Epitope mapping identified 10 phage clones that specifically bound 2 or 3 GP sera, displaying five different peptide sequences that were not streptococcal in origin. These findings suggest that the antigen specificity of the humoral response to BHS in GP does not differ from that of non-psoriatic individuals.

A modular platform for targeted RNAi therapeutics

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 RNAs1–3 (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 targeting4–8, 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.A self-assembled modular siRNA delivery platform enables the construction of a theoretically unlimited repertoire of carriers to target distinct cell surface receptors in the service of personalized medicine.

Isolation of scFvs that Inhibit the NS3 Protease of Hepatitis C Virus by a Combination of Phage Display and a Bacterial Genetic Screen

The need for inhibitors for enzymes linked with microbial infection, specifically the NS3 protease of hepatitis C virus (HCV), inspired us to develop a unique, rapid and easy color-based method described herein. The NS3 serine protease of HCV has a role in processing viral polyprotein and it has been implicated in interactions with various cell constituents, resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for antiviral drugs.We established a genetic screen that is based on coexpression of NS3, a beta-galactosidase reporter that is cleavable by NS3, and potential inhibitors within the same bacterial cell. A single-chain antibody (scFv) library was prepared from spleens of NS3-immunized mice and the screen was used to isolate a panel of protease-inhibiting scFvs. Candidate scFvs were validated for inhibitory activity using an o-nitrophenyl-beta-galactoside (ONPG) hydrolysis assay.The methods can be used more generally to isolate protease-inhibiting cytoplasmic intrabodies able to inhibit proteases or other activities that can be linked with the phenotype of Escherichia coli.

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.