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.

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.

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.

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.

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.

A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia рastoris 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).

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/β-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 HA 306–318 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.

С. С. Терехов, И. В. Смирнов, О. Г. Шамборант, Т. В. Бобик, Д. Г. Илюшин, А. Н. Мурашев, И. А. Дьяченко, В. А. Паликов, В. Д. Кнорре, А. А. Белогуров, Н. А. Пономаренко, Е. С. Кузина, Д. Д. Генкин, P. Masson, А. Г. Габибов Институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова РАН, 117997, Москва, ул. Миклухо-Маклая, 16/10 Филиал Института биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова РАН, 142290, Пущино Московской обл. Казанский федеральный университет, 420000, Казань, ул. Кремлевская, 18 Институт биологии гена РАН, 119334, Москва, ул. Вавилова, 34/5 ОАО «Фармсинтез», 197110, Санкт-Петербург, ул. Красного Курсанта, 25ж *E-mail: sterekhoff@gmail.com Поступила в редакцию 01.09.2015

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.

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.

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.

Рекомбинантные белки стали одними из лидирующих среди современных фармакологических препаратов. Вместе с тем существующие методы анализа фармакокинетики в большинстве своем не учитывают процесс биодеградации белкового препарата. Закономерности биодеградации белкового фармпрепарата позволяют определить основные пути его выведения из организма при проведении доклинических испытаний. Использование флуоресцентных агентов высокой яркости красного и дальне-красного диапазона дает возможность проводить неинвазивные исследования выведения и распределения белковых препаратов в живом организме. Мы предложили использовать метод избыточной конъюгации белков флуорофорами для создания зонда, позволяющего наблюдать биодеградацию рекомбинантной бутирилхолинэстеразы в организме живой мыши. Установлено, что максимум разгорания флуоресценции, отражающей деградацию фермента, наблюдается через 6 ч после внутривенного введения препарата. Показано, что избыточная конъюгация флуорофора приводит к существенному изменению фармакокинетических параметров препарата. Этот факт необходимо учитывать при выборе метода флуоресцентного «биоимиджинга» при проведении фармакокинетических исследований.