Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy.

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

Engineering Artificial Biodiversity of Lantibiotics to Expand Chemical Space of DNA-Encoded Antibiotics.

The discovery of antibiotics was one of the fundamental stages in the development of humanity, leading to a dramatic increase in the life expectancy of millions of people all over the world. The uncontrolled use of antibiotics resulted in the selection of resistant strains of bacteria, limiting the effectiveness of antimicrobial therapy nowadays. Antimicrobial peptides (AMPs) were considered promising candidates for next-generation antibiotics for a long time. However, the practical application of AMPs is restricted by their low therapeutic indices, impaired pharmacokinetics, and pharmacodynamics, which is predetermined by their peptide structure. Nevertheless, the DNA-encoded nature of AMPs enables creating broad repertoires of artificial biodiversity of antibiotics, making them versatile templates for the directed evolution of antibiotic activity. Lantibiotics are a unique class of AMPs with an expanded chemical space. A variety of post-translational modifications, mechanisms of action on bacterial membranes, and DNA-encoded nature make them a convenient molecular template for creating highly representative libraries of antimicrobial compounds. Isolation of new drug candidates from this synthetic biodiversity is extremely attractive but requires high-throughput screening of antibiotic activity. The combination of synthetic biology and ultrahigh-throughput microfluidics allows implementing the concept of directed evolution of lantibiotics for accelerated creation of new promising drug candidates.

Production of Recombinant Protein Self-Assembly Nanocontainers in a Prokaryotic System.

We optimized a method for the preparation and purification of self-assembled protein nanocontainers EPN, the latest achievement in protein engineering. These nanocontainers are highly stable and provide the possibility of highly specific loading of a protein therapeutic drug. The described technique can be proposed as a tool for production of nanocontainers in a prokaryotic system. The obtained nanocontainers for the delivery of protein preparations can be used in the treatment of chronic, autoimmune, and oncological diseases.

Differential Diagnostics of Active Progressing Multiple Sclerosis Using a Fluorescent Biomarker with Resonance Energy Transfer.

Previous data showed that myelin-reactive autoantibodies found in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis recognize and hydrolyze various fragments of myelin basic protein (MBP). Moreover, antibody-mediated cleavage of the encephalithogenic fragment MBP81-103 flanked with two fluorescent proteins can serve as a new biomarker of multiple sclerosis. Here we describe creation of the next generation of this biomarker based on antibody-dependent degradation of a new chemically synthesized fluorescent substrate with resonance energy transfer that contains fluorophore Cy5 and quencher QXL680 separated by MBP81-99 protein (Cy5-MBP81-99-QXL680). This substrate is degraded during incubation with purified antibodies and B cells from patients with multiple sclerosis, but not healthy volunteers.

Application of Tetrameric Recombinant Human Butyrylcholinesterase as a Biopharmaceutical for Amelioration of Symptoms of Acute Organophosphate Poisoning.

We present a procedure for optimizing the expression of recombinant tetrameric butyrylcholinesterase that enables large-scale production with the yield >30 mg/liter (>90 mg/roller bottle). Intravenous injection of the preparation significantly increased survival and decreased the severity of symptoms of poisoning with paraoxon, an organophosphorus toxin.

[Expression Of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris].

A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day).

Creation of Recombinant Biocontrol Agents by Genetic Programming of Yeast.

Bacterial infections caused by antibiotic-resistant pathogens pose an extremely serious and elusive problem in healthcare. The discovery and targeted creation of new antibiotics are today among the most important public health issues. Antibiotics based on antimicrobial peptides (AMPs) are of particular interest due to their genetically encoded nature. A distinct advantage of most AMPs is their direct mechanism of action that is mediated by their membranolytic properties. The low rate of emergence of antibiotic resistance associated with the killing mechanism of action of AMPs attracts heightened attention to this field. Recombinant technologies enable the creation of genetically programmable AMP producers for large-scale generation of recombinant AMPs (rAMPs) or the creation of rAMP-producing biocontrol agents. The methylotrophic yeast Pichia pastoris was genetically modified for the secreted production of rAMP. Constitutive expression of the sequence encoding the mature AMP protegrin-1 provided the yeast strain that effectively inhibits the growth of target gram-positive and gram-negative bacteria. An antimicrobial effect was also observed in the microculture when a yeast rAMP producer and a reporter bacterium were co-encapsulated in droplets of microfluidic double emulsion. The heterologous production of rAMPs opens up new avenues for creating effective biocontrol agents and screening antimicrobial activity using ultrahigh-throughput technologies.

B Cell Profiling in Patients with Pemphigus Vulgaris.

Pemphigus vulgaris is a severe, socially significant autoimmune disease associated with autoantibodies to the desmoglein 3 antigen. The disease affects all age groups, beginning at 18 years of age; the mortality rate of pemphigus can reach as high as 50%, depending on a patient's age and a number of other factors. There is no highly selective or personalized therapy for pemphigus vulgaris at the moment. One of the well-known therapeutic approaches to the disease is to use rituximab, an anti-CD20 antibody that can help achieve B cell depletion in peripheral blood. To solve the problem of nonspecific elimination of B cells in patients with pemphigus vulgaris, it is reasonable to use specific immunoligands, their choice being based on an assessment of the level of autoantibodies specific to each of the fragments of desmoglein. In this work, the proportion of autoreactive B cells in patients diagnosed with pemphigus vulgaris is found to be 0.09-0.16%; a positive correlation was revealed between the antibody level and the number of autoreactive B cells to various fragments of desmoglein.

High-Throughput Screening of Biodiversity for Antibiotic Discovery.

The increasing number of infections caused by antibiotic-resistant strains of pathogens challenges modern technologies of drug discovery. Combinatorial chemistry approaches are based on chemical libraries. They enable the creation of high-affinity low-molecular-weight ligands of the therapeutically significant molecular targets of human cells, thus opening an avenue toward a directed design of highly effective therapeutic agents. Nevertheless, these approaches face insurmountable difficulties in antibiotic discovery. Natural compounds that have evolved for such important characteristics as broad specificity and efficiency are a good alternative to chemical libraries. However, unrestricted use of natural antibiotics and their analogues leads to avalanche-like spread of resistance among bacteria. The search for new natural antibiotics, in its turn, is extremely complicated nowadays by the problem of antibiotic rediscovery. This calls for the application of alternative high-throughput platforms for antibiotic activity screening, cultivation of "unculturable" microorganisms, exploration of novel antibiotic biosynthetic gene clusters, as well as their activation and heterologous expression. Microfluidic technologies for the screening of antibiotic activity at the level of single cells are, therefore, of great interest, since they enable the use of a single platform to combine the technology of ultrahigh-throughput screening, next-generation sequencing, and genome mining, thus opening up unique opportunities for antibiotic discovery.

Mouse Model for Assessing the Subchronic Toxicity of Organophosphate Pesticides.

The development of antidotes to organophosphate poisons is an important aspect of modern pharmacology. Recombinant acetylcholinesterase and butyrylcholinesterase are effective DNA-encoded acceptors of organophosphate poisons and, in particular, pesticides. Here, we present the results of a study on the effectiveness of recombinant butyrylcholinesterase (BChE) in modeling organophosphate poisoning caused by oral administration of paraoxon at a dose of 2 mg / kg. The study showed a high activity of BChE as a protective agent for subchronic anticholinesterase poisoning in an in vivo model. The administration of BChE in a dose of 20 mg / kg allows one to avoid mortality, and also contributed to rapid recovery after model poisoning.

Chemical Polysialylation and In Vivo Tetramerization Improve Pharmacokinetic Characteristics of Recombinant Human Butyrylcholinesterase-Based Bioscavengers.

Organophosphate toxins (OPs) are the most toxic low-molecular compounds. The extremely potent toxicity of OPs is determined by their specificity toward the nerve system. Human butyrylcholinesterase (hBChE) is a natural bioscavenger against a broad spectrum of OPs, which makes it a promising candidate for the development of DNA-encoded bioscavengers. The high values of the protective index observed for recombinant hBChE (rhBChE) make it appropriate for therapy against OP poisoning, especially in the case of highly toxic warfare nerve agents. Nevertheless, large-scale application of biopharmaceuticals based on hBChE is restricted due to its high cost and extremely rapid elimination from the bloodstream. In the present study, we examine two approaches for long-acting rhBChE production: I) chemical polysialylation and II) in-vivo tetramerization. We demonstrate that both approaches significantly improve the pharmacokinetic characteristics of rhBChE (more than 5 and 10 times, respectively), which makes it possible to use rhBChE conjugated with polysialic acids (rhBChE-CAO) and tetrameric rhBChE (4rhBChE) in the treatment of OP poisonings.

Excessive Labeling Technique Provides a Highly Sensitive Fluorescent Probe for Real-time Monitoring of Biodegradation of Biopolymer Pharmaceuticals in vivo.

Recombinant proteins represent a large sector of the biopharma market. Determination of the main elimination pathways raises the opportunities to significantly increase their half-lives in vivo. However, evaluation of biodegradation of pharmaceutical biopolymers performed in the course of pre-clinical studies is frequently complicated. Noninvasive pharmacokinetic and biodistribution studies in living organism are possible using proteins conjugated with near-infrared dyes. In the present study we designed a highly efficient probe based on fluorescent dye self-quenching for monitoring of in vivo biodegradation of recombinant human butyrylcholinesterase. The maximum enhancement of integral fluorescence in response to degradation of an intravenously administered enzyme was observed 6 h after injection. Importantly, excessive butyrylcholinesterase labeling with fluorescent dye results in significant changes in the pharmacokinetic properties of the obtained conjugate. This fact must be taken into consideration during future pharmacokinetic studies using in vivo bioimaging.