BACKGROUND:The biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production.RESULTS:We have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated. The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs. Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

The book examines the ‘poetics of joy’ in Dante’s Divine Comedy across the disciplines of lexicography, language history, philology, and theology, with reliance on an extensive body of research and supplemented with numerous comments to the poem. The book consists of two parts. In the first, the author analyses the vocabulary used by Dante and other medieval authors to express the notion. Dante’s artistic world exists within a broader cultural and religious context of the period. The book’s second part is devoted to the means by which the vocabulary denoting joy is rendered in M. Lozinsky’s now classical translation of the Comedy. The addendum contains the author’s own translations of several cantos from the Paradiso as well as of Jacopone da Todi’s lauda on the meaning of joy. The book will certainly interest Dante scholars and a broader audience of readers who are keen to understand the Divine Comedy — a unique work of world literature that has roused profound curiosity for more than seven centuries.

The development of new therapies for malignant tumors is an urgent task. Currently, the humanized antibody trastuzumab is considered the "gold standard" in the complex treatment of breast tumors with overexpression of HER2, human epidermal growth factor receptor 2. However, in some cases, resistance to the specified preparation is observed. The search for new therapies for HER2-associated tumors seems to be an important area of research. A number of clinical studies are currently underway on the use of human interferon-beta (IFN-beta) in oncology. Most of these studies use viral vectors carrying the interferon-beta gene to reduce the systemic effect of this cytokine. The immunocytokine complex of the bispecific antibody and IFN-beta we developed can also avoid the systemic action of IFN-beta. Part of the development of such a complex is the creation of bispecific antibodies of various formats. Based on the neutralizing B16 antibody to IFN-beta and the trastuzumab (Tz) antibody specific for the HER2 receptor, we obtained various variants of bispecific antibodies in Fab-scFv format. It was shown that the proteins obtained bind and neutralize IFN-beta, and they also bind the HER2 receptor in tumor cell lysates and as a recombinant extracellular domain. Such molecules in the immunocytokine complex can be used as delivery vehicles of IFN-beta to HER2-positive tumor cells.

We obtained dimeric forms of IgA1- and IgA2m1-isotypes of FI6 antibody broadly specific to hemagglutinins of different subtypes of influenza A virus. It was shown that the dimers of IgA1 isotype are characterized by a higher antigen-binding activity compared to the IgA2m1 dimers. The affinity of IgA1 dimers to the strains of the H1N1 subtype is higher than that of the H3N2 subtype, which correlates with the properties of the parental human FI6 antibody.

We obtained recombinant variants of human antibody FI6 broadly specific to hemagglutinins of the influenza A virus. On the basis of a bi-promoter (CMV, hEF1-HTLV) vector, we developed genetic constructs for the expression of the heavy and light chains of the immunoglobulins of IgA1-, IgA2m1-, and IgG-isotypes. Following transfection and selection, stable Chinese hamster ovary (CHO) cell lines were produced. The antibodies of IgA1-, IgA2m1-, and IgG-isotypes were purified from culture media. We performed an immunochemical characterization and studied their interactions with influenza A strains of the H1N1- and H3N2-subtypes. It was shown that recombinant FI6 variants of the IgA-isotype retain the properties of the parental IgG antibody to demonstrate specificity to all the strains tested. The strongest binding was observed for the H1N1 subtype, which belongs to hemagglutinins of phylogenetic group I.

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

Currently, there are no approved therapies for targeted prevention and treatment of Ebola hemorrhagic fever. In the present work, we describe the development of a eukaryotic expression system for the production of three full-length chimeric antibodies (IgG1-kappa isotypes) GPE118, GPE325, and GPE534 to the recombinant glycoprotein of the Ebola virus (EBOV GP), which is a key factor in the pathogenicity of the disease. The immunochemical properties of the obtained antibodies were studied by immunoblotting and indirect, direct, and competitive ELISA using the recombinant EBOV proteins rGPdTM, NP, and VP40. The authenticity of the antibodies and the absence of cross-specificity with respect to the structural proteins NP and VP40 of the Ebola virus were proved. The epitope specificity of the resulting recombinant antibodies was studied using commercial neutralizing antibodies against the viral glycoprotein. The recombinant antibodies GPE118, GPE325, and GPE534 were shown to recognize glycoprotein epitopes that coincide or overlap with the epitopes of three well-studied neutralizing anti-Ebola virus antibodies.

Production and application of therapeutic monoclonal antibodies are second only to vaccines in the world pharmaceutical market. The most common therapeutic antibodies are monoclonal antibodies (mAbs) of the IgG isotype that are produced in eukaryotic CHO cells. In recent years, there has been a considerable interest in developing treatment medications based on IgA antibodies, which can have a wide range of effector functions on human mucous membranes. To study the expression level of immunoglobulin A (IgA) in mammal cells, we designed a set of bipromoter (CMV and EF1α) vectors. The vectors contain gene fragments that encode the heavy chain variable domain (VH) and the light chain variable domain (VL) of the human monoclonal antibody FI6v3 against the hemagglutinin of influenza virus A. They also contain gene fragments that encode the light chain (kappa type) constant domain and the heavy chain constant domain of the human antibody IgA1. The expression vectors differed in the orientation of the promoters and the presence or absence of introns. Two variants of the full-length light and heavy chains were cloned into a eukaryotic expression vector in head-to-head and head-to-tail orientations. The resulting plasmids were transfected into CHO-DG44 and HEK-293T cells. The antibody expression level for the stable transfection of CHO-DG44 and HEK-293T cell cultures was determined by ELISA. The results of the experiments showed that the expression of FI6v3-IgA1 antibodies significantly increased when eukaryotic cells were transfected with the plasmid pBiPr-ABIgA1FI6-Iht in which the heavy chain of IgA1 contains introns and the promoters are arranged head-to-tail.

Production and application of therapeutic monoclonal antibodies take second place in the global pharmaceutical market after vaccines. Nowadays the IgG1 is one of the prevailing monoclonal antibodies type for the therapeutic use that are produced in CHO cells. Recently the possibility of developing therapeutic drugs based on antibodies of IgA-isotype has demonstrated a broad range of effector functions on human mucous membranes, which has caused a significant interest in this research field. In our research about level of IgA1 antibody expression in mammal cells, we designed a set of bipromoter (CMV, EF1α) vectors containing variable regions of heavy chain (VH) and light chain (VL) genes from human monoclonal antibody FI6v3 against influenza A virus, constant regions IgA1 of heavy and light chains of human antibody (with or without introns). The vectors differed in mutual orientation of promoters and presence or lack of introns. Two versions of the full-length light and heavy chains were cloned in eukaryotic expressing vector in “head-head”/ “head-tail” orientations and then transfected into HEK-293T and CHO/dhfr-cells. The level of antibody production was determined using ELISA test for transfection of CHO DG44 and HEK-293T cell culture. The results of the experiments showed a significant increase in the production of FI6v3-IgA1 antibodies when eukaryotic cells were transfected with a plasmid pBiPr-ABIgA1FI6-Iht containing introns in constant regions of IgA1 and with orientation of transcription in the “head-tail” direction.

We studied the possibility of using a broadly neutralizing anti-influenza A antibody as a module for the development of different protein constructs for diagnostics. For this purpose, we constructed two recombinant proteins—a Fab-fragment of the antibody and Fab-mCherry, which is a hybrid of the Fab-fragment and the mCherry fluorescent protein. Both proteins were expressed in Escherichia coli cells and purified in a functionally active state from culture medium. The antibody Fab-fragment was shown to bind all 11 tested strains of the influenza A H1N1 and H3N2 subtypes. A stronger binding was observed for group I hemagglutinins; this correlates with the immunochemical profile of the parental antibody. Comparison of the dissociation constants of complexes of the antibody Fab-fragment and Fab-mCherry with A(H1N1)/Solomon Islands/03/06 virus particles demonstrated that the attachment of the mCherry protein did not interfere with the antigen-binding properties of the antibody Fab-fragment.

We studied the possibility of using a broadly neutralizing anti-influenza A antibody as a module for the development of different protein constructs for diagnostics. For this purpose we constructed two recombinant proteins — an antibody Fab-fragment and Fab-mCherry, which is a hybrid of the Fab-fragment and a fluorescent protein mCherry. Both proteins were expressed in Escherichia coli cells and purified in a functionally active state from cultivation medium. The antibody Fab-fragment was shown to bind all eleven tested strains of the influenza A H1N1 and H3N2 subtypes. The stronger binding was observed for the group I hemagglutinins that correlates with the immunochemical profile of the parental antibody. Comparison of the dissociation constants of complexes of the antibody Fab-fragment and Fab-mCherry with A(H1N1)/Solomon Islands/03/06 virus particles demonstrated that the attachment of mCherry protein did not interfere with the antigen-binding properties of the antibody Fab-fragment.

The development of HIV vaccine remains an important goal in prophylaxis and therapy of HIV/ AIDS epidemics. There are various approaches for development of а candidate vaccine based on induction of neutralizing antibodies and cell-mediated immunity. Synthetic peptides are considered promising vaccine antigens since they are capable of activating both humoral and cellular immune response. HIV-1 envelope gp120 is the target for neutralizing antiviral antibodies. The V3 region of the HIV-1 gp120 is highly immunogenic and important for the virus-coreceptor interaction. In a RV144 vaccine trial, the levels of vaccine-induced IgG antibodies recognizing V1V2 regions from multiple HIV-1 subtypes show inverse correlations with a risk for HIV-1 infection. Meanwhile, HIV is characterized by high diversity. The consensus and mosaic immunogens are complete but artificial proteins, which are computationally designed to elicit immune responses with improved cross-reactive broadness. We have been studied immunogenic properties of synthetic peptides derived from V1, V2, V3 loop regions of the consensus M HIV1 (CON-S) sequence group of the gp 120 envelope protein and V3 loop derived from a Russian RUA022a2 isolate. These peptides specifically reacted to HIV-positive sera in ELISA, thus indicating their similarity to appropriate HIV proteins. The peptides proved to be weakly immunogenic. Therefore, Freund complete adjuvant was used to enhance peptide immunogenicity. To assess the immunogenicity, the mice were immunized with a peptide mixture. Antibodies have been developed to every peptide from the mixture, being, predominantly, of IgG isotype. The antibody titers depended on the length of peptide sequences. However, the sera from immunized mice did not have a HIV neutralizing activity. The serum neutralization was assessed by pseudovirus-based assay, using a molecular clone of virus isolates CAP 45.2.00.G3 and QH.209.14.M.EnvA2. The virus neutralization is a complex process and may be influenced by several factors, such as antibody titer, isotype, or antibody structure. Probably, to induce neutralizing antibodies by this peptide mixture, it is necessary to choose appropriate adjutants and immunization schedule. Moreover, it was shown that peptides could increase in vitro virus infectivity in pseudovirus-based model, using the CAP 45.2.00.G3, QH209.14M.ENV.A2, QD435.100M.ENV.E1 molecular clone. These viral isolates belong to different HIV-1 subtypes.

We have studied the possibility to increase the production level of recombinant antibodies in CHO cells by using the addition of several components into cultural medium. Using the transient expression culture, it was shown that the addition of the sodium salt of valproic acid results in 20% increase of expression yield. The most prominent effect is observed when 100 mM zinc sulfate is added. Upon cultivation of stable cell line the reduction of L-glutamine concentration to 0.2 mM gives rise to increased culture viability and the growth in productivity by a factor of 2.5. The cultivation at low L-glutamine concentration can lead to higher cell density and prolonged cultivation, which is very important for scaled-up cell fermentation in spinner flasks and bioreactors.