Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1.

Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.

Evolution of catalytic centers of antibodies by virtual screening of broad repertoire of mutants using supercomputer.

It is proposed to perform quantum mechanical/molecular dynamics calculations of chemical reactions that are planned to be catalyzed by antibodies and then conduct a virtual screening of the library of potential antibody mutants to select an optimal biocatalyst. We tested the effectiveness of this approach by the example of hydrolysis of organophosphorus toxicant paraoxon using kinetic approaches and X-ray analysis of the antibody biocatalyst designed de novo.

Preparation of Recombinant Serpin from Red King Crab Paralithodes сamtschaticus for Biomedical Research Purposes.

Genetic constructs with different leader sequences for intra- and extracellular expression of the target protein were generated and an original method for effective selection of clones with maximum expression was developed. For intracellular expression in the Pichia pastoris system, seprin content in cells was 6 mg/liter.

Genetic Engineering of Native Chain Combinations of B-Cell Repertoires on the Surface of Methylotrophic Yeasts Pichia pastoris.

We designed genetic constructs for exposing Fab-fragment library of natively paired single cell B-cell receptors on the surface of Pichia pastoris yeast cells. We have previously obtained the A17 antibody in our laboratory [6]. In this study we showed that the newly designed genetic constructs provide a compatible level of A17 antibody Fab fragment on the surface of yeast cells as well as in the case of vectors containing DNA fragments corresponding to each chain of the antibody. The data suggest that the developed approach for constructing immunoglobulin gene libraries is adequate and fully convenient for studying properties of the real human B-lymphocyte repertoire.

Design of Chemical Conjugate for Targeted Therapy of Multiple Sclerosis Based of Constant Fragment of Human Antibody Heavy Chain and Peptoid Analog of Autoantigen MOG35-55.

Elimination of B cells producing autoantibodies to neuroantigens is considered as beneficial in the treatment of multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG) is a significant autoantigen in multiple sclerosis. It was shown that MOG-like peptoid AMogP3 can bind autoantibodies produced by pathological lymphocytes. We propose a structure of an innovative drug for targeted elimination of the pool of autoreactive B cells responsible for multiple sclerosis pathogenesis; this compound is a complex of peptoid AMogP3 with Fc fragment of human immunoglobulin. The obtained Fc-PEG-AMogP3 conjugate effectively interact with autoreactive antibodies, which attests to their high therapeutic potential.

Heterodimer HLA-DM Fused with Constant Fragment of the Heavy Chain of the Human Immunoglobulin Accelerates Influenza Hemagglutinin HA306-318 Loading to HLA-DR1.

Major histocompatibility complex class II (MHC II) plays an important role not only in the adaptive immune responses to foreign pathogens, but also in the development of some autoimmune diseases. Non-classical MHC, HLA-DM is directly involved in MHC II loading with the peptide. To study this process, we synthesized recombinant proteins HLA-DR1 and HLA-DM. α/ß-Chains of DR1 heterodimer contained C-terminal leucine domains of the fos and jun factors, respectively. Each DM chain contained constant fragment of human antibody heavy chain fused via a long linker domain. In addition, DM α-chain carried N165D substitution suppressing potential glycosylation at this site. We observed significant acceleration of DR1 peptide loading with influenza HA306-318 hemagglutinin in the presence of DM, which indicates functionality of recombinant DR1-DM protein couple. Our results can be used to study the presentation of other viral and self-antigens and can become the basis for the development of new drug modeling.

[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).

Specific Depletion of Myelin-Reactive B Cells via BCR-Targeting.

B cells play a crucial role in the development and pathogenesis of systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce antibodies, but also secrete pro-inflammatory cytokines and present specific autoantigens to T cells. The treatment of autoimmune diseases via the elimination of the majority of B cells using the monoclonal anti-CD19/20 antibody (Rituximab) causes systemic side effects and, thus, requires a major revision. Therapeutic intervention directed towards selective elimination of pathogenic autoreactive B cells has the potential to become a universal approach to the treatment of various autoimmune abnormalities. Here, we developed a recombinant immunotoxin based on the immunodominant peptide of the myelin basic protein (MBP), fused to the antibody Fc domain. We showed that the obtained immunotoxin provides selective in vivo elimination of autoreactive B cells in mice with experimental autoimmune encephalomyelitis. The proposed conception may be further used for the development of new therapeutics for a targeted treatment of multiple sclerosis and other autoimmune disorders.

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.

Recombinant human butyrylcholinesterase as a new-age bioscavenger drug: development of the expression system.

Butyrylcholinesterase (BChE) is a serine hydrolase (EC 3.1.1.8) which can be found in most animal tissues. This enzyme has a broad spectrum of efficacy against organophosphorus compounds, which makes it a prime candidate for the role of stoichiometric bioscavenger. Development of a new-age DNA-encoded bioscavenger is a vival task. Several transgenic expression systems of human BChE were developed over the past 20 years; however, none of them has been shown to make economic sense or has been approved for administration to humans. In this study, a CHO-based expression system was redesigned, resulting in a significant increase in the production level of functional recombinant human butyrylcholinesterase as compared to the hitherto existing systems. The recombinant enzyme was characterized with Elman and ELISA methods.

Polyreactive monoclonal autoantibodies in multiple sclerosis: functional selection from phage display library and characterization by deep sequencing analysis.

Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system that primarily affects young and middle-aged people. It is widely accepted that B lymphocyte activation is required for MS progression. Despite the fact that the exact triggering mechanisms of MS remain enigmatic, one may suggest that MS can be induced by viral or bacterial infection in combination with specific genetic and environmental factors. Using deep sequencing and functional selection methodologies we characterized clones of poly- and cross-reactive antibodies that are capable of simultaneous recognition of viral proteins and autoantigens. The latter, in turn, possibly may trigger MS progression through molecular mimicry. It was identified that two cross-reactive antigens are probably recognized by light or heavy chains individually. According to the high structural homology between selected autoantibodies and a number of various antiviral IgGs, we suggest that a wide range of pathogens, instead of a single virus, be regarded as possible triggers of MS.

Creation of catalytic antibodies metabolizing organophosphate compounds.

Development of new ways of creating catalytic antibodies possessing defined substrate specificity towards artificial substrates has important fundamental and practical aspects. Low immunogenicity combined with high stability of immunoglobulins in the blood stream makes abzymes potent remedies. A good example is the cocaine-hydrolyzing antibody that has successfully passed clinical trials. Creation of an effective antidote against organophosphate compounds, which are very toxic substances, is a very realistic goal. The most promising antidotes are based on cholinesterases. These antidotes are now expensive, and their production methods are inefficient. Recombinant antibodies are widely applied in clinics and have some advantage compared to enzymatic drugs. A new potential abzyme antidote will combine effective catalysis comparable to enzymes with high stability and the ability to switch on effector mechanisms specific for antibodies. Examples of abzymes metabolizing organophosphate substrates are discussed in this review.

[Therapeutic effect of encapsulated into the nanocontainers MBP immunodominant peptides on EAE development in DA rats].

Multiple Sclerosis (MS) is a serve autoimmune neurodegenerative disease. Development of innovative approaches of MS treatment is of a high priority in the modern immunology and pharmacy. In the present study we showed high therapeutic efficiency of immunodominant peptides of myelin basic protein (MBP) incorporated into the monolayer mannosylated liposomes on the development of experimental autoimmune encephalomyelitis (EAE) in DA rats. MBP is a component ofoligodendrocytes' membrane, which form axonal sheath, and is one of the major autoantigens in MS. We analyzed binding pattern ofanti-MBP autoantibodies from MS patients using previously designed MBP epitope library. Utilizing the same approach we investigated pool of anti-MBP antibodies from SJL/J and C57/BL6 mice and DA rats with induced EAE. The most relevant rodent model to MS was EAE in DA rats according to the autoantibodies' binding pattern. We selected three immunodominant MBP fragments encapsulated in monolayer mannosylated liposomes for the following treatment of verified DA rodent model. MBP fragment 46-62 was the most effective in reducing of the first EAE attack, whereas MBP 124-139 and 147-160 inhibited development of pathology during remission stage. Simultaneous administration of these peptides in liposomes significantly decreased level of anti-MBP antibodies. Synergetic therapeutic effect of MBP fragments reduced integral disease score by inhibiting first EAE wave and subsequent remission, thus, our findings disclosure novel approaches for efficient treatment of Multiple Sclerosis.

[Expression of the catalytic antibodies in eukaryotic systems].

Expression of recombinant antibodies in mammalian cells is one of key problems in immunobiotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively done in yeast cells. We obtained expression strains of the methylotrophic beast Pichia pastoris producing single chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab) and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant Kd and the first order constant (k2) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (Phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and single chain antibody A.17 expressed in CHO and E. coli cells respectively.

[Functional degradation of myelin basic protein. Proteomic approach].

Proteolytic degradation of autoantigens is of prime importance in current biochemistry and immunology. The most fundamental issue in this field is the functional role of peptides produced when the specificity of hydrolysis changes during the shift from health to disease and from normal state to pathology. The identification of specific peptide fragments in many cases proposes the diagnostic and prognostic criterion in the pathology progression. The aim of this work is comparative study of the degradation peculiarities of one of the main neuroantigen, myelin basic protein by proteases, activated during progress of pathological demyelinating process, and by proteasome of different origin. The comparison of specificity of different studied biocatalysts gives reason to discuss the critical change in the set of myelin basic protein fragments capable to be presented by major histocompatibility complex class I during neurodegeneration, which can promote the progress of autoimmune pathological process.

[Diagnostic and pathogenetic implications of the site specificity of antibody proteases in multiple sclerosis].

Disseminated sclerosis is currently regarded as a CNS autoimmune disease. One of the mechanisms behind this pathology is antibody (AB) formation. In this context, recent data on AB with proteolytic activity are of importance because they participate in selective proteolysis of myelin proteins in patients with disseminated sclerosis. This paper focuses on AB-proteases associated with disseminated sclerosis and site-specificity of antibody-mediated proteolysis of myelin basic protein. Protocol of serodiagnostic algorithm to be used in clinical practice is described.

Suppression of ongoing experimental allergic encephalomyelitis in DA rats by novel peptide drug, structural part of human myelin basic protein 46-62.

Previously, we demonstrated that autoantibodies (AAb) in multiple sclerosis (MS) reveal site-specific binding and cleavage toward myelin basic protein (MBP) epitope library. We have found several fragments of MBP immunodominant in terms of AAb binding. Here, we applied these peptides to DA rats with induced protracted relapsing experimental allergic encephalomyelitis (EAE) most closely related to MS. DA rats with EAE induced by syngenic spinal cord homogenate in complete Freund's adjuvant were treated by nasal route with human MBP 46-62, 81-102, 124-139, 147-170, and Copaxone. MBP 124-139 and 147-170 displayed only mild therapeutic effects but MBP 46-62 significantly reduced EAE, reflected by lower clinical scores and shorter EAE duration compared to controls.