Synthetic antibody discovery against native antigens by CRISPR/Cas9-library generation and endoplasmic reticulum screening.

Despite the significant advances of antibodies as therapeutic agents, there is still much room for improvement concerning the discovery of these macromolecules. Here, we present a new synthetic cell-based strategy that takes advantage of eukaryotic cell biology to produce highly diverse antibody libraries and, simultaneously, link them to a high-throughput selection mechanism, replicating B cell diversification mechanisms. The interference of site-specific recognition by CRISPR/Cas9 with error-prone DNA repair mechanisms was explored for the generation of diversity, in a cell population containing a gene for a light chain antibody fragment. We achieved up to 93% of cells containing a mutated antibody gene after diversification mechanisms, specifically inside one of the antigen-binding sites. This targeted variability strategy was then integrated into an intracellular selection mechanism. By fusing the antibody with a KDEL retention signal, the interaction of antibodies and native membrane antigens occurs inside the endoplasmic reticulum during the process of protein secretion, enabling the detection of high-quality leads for expression and affinity by flow cytometry. We successfully obtained antibody lead candidates against CD3 as proof of concept. In summary, we developed a novel antibody discovery platform against native antigens by endoplasmic synthetic library generation using CRISPR/Cas9, which will contribute to a faster discovery of new biotherapeutic molecules, reducing the time-to-market.

Quality and safety requirements for sustainable phage therapy products.

The worldwide antibiotic crisis has led to a renewed interest in phage therapy. Since time immemorial phages control bacterial populations on Earth. Potent lytic phages against bacterial pathogens can be isolated from the environment or selected from a collection in a matter of days. In addition, phages have the capacity to rapidly overcome bacterial resistances, which will inevitably emerge. To maximally exploit these advantage phages have over conventional drugs such as antibiotics, it is important that sustainable phage products are not submitted to the conventional long medicinal product development and licensing pathway. There is a need for an adapted framework, including realistic production and quality and safety requirements, that allows a timely supplying of phage therapy products for 'personalized therapy' or for public health or medical emergencies. This paper enumerates all phage therapy product related quality and safety risks known to the authors, as well as the tests that can be performed to minimize these risks, only to the extent needed to protect the patients and to allow and advance responsible phage therapy and research.

Toxicological evaluation of Alpinia oxyphylla-derived molecule (PD-00105): In vitro genotoxicity studies and 90-day oral toxicity study in rats.

PD-00105 corresponds to a compound initially identified in the fruit of Alpinia oxyphylla Miq., obtained by chemical synthesis and proposed to be use in dietary supplements for its potential neuroprotective properties. The aim of this study was to perform a toxicological evaluation of PD-00105 in accordance with the testing strategy recommended by food regulatory authorities. All studies were conducted in accordance with Good Laboratory Practice (GLP), and followed the Organization for Economic Co-operation and Development (OECD) test guidelines for chemicals. Studies included a bacterial reverse mutation test, one in vitro micronucleus test in mammalian cells, and a repeated dose 90-day oral toxicity study. No sign of toxicity was observed in the two genotoxicity tests. The test item induced a significant liver and kidney toxicity at high doses (50 and 100 mg/kg BW/day), highlighted by significant increases in liver and kidney absolute and relative weights, associated with histopathological findings and concomitant changes in hematology and clinical chemistry. Increases in alanine aminotransferase, alkaline phosphatase, total protein, albumin, globulin, cholesterol, LDL, and HDL have been measured in these two groups. However, findings observed in the low-dose group (10 mg/kg BW/day) were considered as minimal and non-adverse, and were limited to an increase in liver weight in males and in kidneys weight in females, without concomitant changes in blood chemistry. The No Observed Adverse Effect Level (NOAEL) of PD-00105 was established as 10 mg/kg BW/day under the conditions of this study. This study substantiates the use of PD-00105 in dietary supplements at doses of 10 mg/day, taking into account a safety margin factor for dose conversion to humans.

Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds.

Chronic wounds that fail to heal are a common complication of diabetes mellitus and the most common precipitating reason for nontraumatic lower limb amputation. Unfortunately, the bacterial species that cause these infections are becoming more resistant to antibiotics, making them increasingly difficult to treat. We assessed the feasibility of combating chronic bacterial infections with a topically delivered bacteriophage cocktail in two animal models of diabetes mellitus. Microbiological, planimetric, and histological parameters were compared in debrided infected wounds with or without topical bacteriophage treatment. We determined that bacteriophage treatment effectively decreased bacterial colony counts and improved wound healing, as indicated by smaller epithelial and dermal gaps, in Staphylococcus aureus and Pseudomonas aeruginosa infections but was not as effective against Acinetobacter baumannii. Although the improvements were more significant in the rodent model than in the porcine model, our results suggest that topically administered bacteriophage treatment may be effective in resolving chronic infections, especially when applied in conjunction with wound debridement. These findings have important implications for the feasibility of using topical antimicrobial therapies to safely treat chronic infections in diabetes mellitus patients.

Author Correction: A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A zebrafish drug screening platform boosts the discovery of novel therapeutics for spinal cord injury in mammals.

Spinal cord injury (SCI) is a complex condition, with limited therapeutic options, that results in sensory and motor disabilities. To boost discovery of novel therapeutics, we designed a simple and efficient drug screening platform. This innovative approach allows to determine locomotor rescue properties of small molecules in a zebrafish (Danio rerio) larval spinal cord transection model. We validated our screening platform by showing that Riluzole and Minocycline, two molecules that are in clinical trials for SCI, promote rescue of the locomotor function of the transected larvae. Further validation of the platform was obtained through the blind identification of D-Cycloserine, a molecule scheduled to enter phase IV clinical trials for SCI. Importantly, we identified Tranexamic acid and further showed that this molecule maintains its locomotor recovery properties in a rodent female contusion model. Our screening platform, combined with drug repurposing, promises to propel the rapid translation of novel therapeutics to improve SCI recovery in humans.

Recombinant antibodies as therapeutic agents: pathways for modeling new biodrugs.

Hybridoma fusion technology, proposed by Köhler and Milstein in 1975, started major developments in the field of monoclonal antibodies (mAbs). During the following 2 decades, their high potential as laboratory tools was rapidly exploited for biotechnology and biomedical applications. Today, mAbs represent over 30% of all biological proteins undergoing clinical trials and are the second largest class of biodrugs after vaccines. With the help of antibody engineering, mAbs have been reduced in size, rebuilt into multivalent molecules, and conjugated with drugs, toxins, or radioisotopes for the treatment of cancer, autoimmune disorders, graft rejection, and infectious diseases. Additionally, in the past few years, important advances have been made in the design, selection, and production of these new types of engineered antibodies. The present review focuses on the structural and functional characteristics of mAbs and their fragments, and also provides a walk through the most important methods used in antibody selection. In addition, the recent trends in antibody engineering for improving antibody clinical efficacy are also reviewed.

Cell type-specific targeting with sindbis pseudotyped lentiviral vectors displaying anti-CCR5 single-chain antibodies.

Lentiviral vectors are among the most efficient tools for gene delivery into mammalian cells. A major goal of lentiviral gene delivery systems is to develop vectors that can efficiently target specific cell types. In the present work, we attempt to generate viral particles for targeting gene delivery. We have used CCR5-positive cells as the target for our strategy. Therefore, we developed a novel Sindbis pseudotyped lentiviral vector where the Sindbis receptor binding envelope protein was modified to directly encode a single-chain antibody fragment (scFv) against the CCR5 chemokine receptor. We have generated two chimeric scFv-Sindbis envelopes, varying the length of the peptide linker that connects the heavy chain and light chain of anti-CCR5 scFv. The two chimeric scFv-Sindbis envelopes were successfully incorporated into lentiviral-derived vectors, and the resulting pseudotyped viral particles showed specific targeting to CCR5-expressing cells. However, our data demonstrate that the length of the peptide linker significantly affects the efficiency of infection. Pseudotyped viral particles, which display single-chain antibody fragments with longer peptide linkers, allowed higher titers of infection. The present study can be a model strategy for specific gene delivery mediated by lentiviral vectors pseudotyped with Sindbis envelope displaying scFv that recognizes specific cellular surface proteins. Furthermore, this strategy has the potential to become a powerful approach for targeting gene delivery in anti- HIV gene therapy due to the important role of CCR5 expression in disease progression.

Intrabodies targeting the Kaposi sarcoma-associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells.

Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen-1 (LANA1) is essential for the maintenance and segregation of viral episomes in KSHV latently infected B cells. We report development of intracellular, rabbit-derived antibodies generated by phage display technology, which bind to N-terminal LANA1 epitopes and neutralize the chromosome-binding activity of LANA1. Although these cloned single-chain variable fragments (scFvs) show relatively low binding affinities for the LANA1 viral antigen in in vitro assays, they nonetheless outcompete KSHV-seropositive human sera for LANA1 epitope binding. In heterologous cells, intracellular intrabody expression inhibits LANA1-dependent plasmid maintenance of both an artificial plasmid containing KSHV LANA1 binding sequences and a bacterial artificial chromosome containing the entire KSHV genome. In KSHV naturally infected primary effusion lymphoma cells, intracellular intrabody expression causes a reduction or loss of the typical LANA1 punctate, nuclear pattern. This morphologically apparent LANA1 dispersion correlates to loss of viral episome by molecular analysis. These data suggest a novel approach to antiherpes viral therapy and confirm LANA1 is critical target for neutralization of KSHV viral latency.