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.

Anti-tumor properties of several cytokines have already been investigated in multiple experiments and clinical trials. However, those studies evidenced substantial toxicities, even at low cytokine doses, and the lack of tumor specificity. These factors significantly limit clinical applications. Due to their high specificity and affinity, tumor-specific monoclonal antibodies or their antigen-binding fragments are capable of delivering fused cytokines to tumors and, therefore, of decreasing the number and severity of side effects, as well as of enhancing the therapeutic index. The present review surveys the actual antibody–cytokine fusion protein (immunocytokine) formats, their targets, mechanisms of action, and anti-tumor and other biological effects. Special attention is paid to the formats designed to prevent the off-target cytokine–receptor interactions, potentially inducing side effects. Here, we describe preclinical and clinical data and the efficacy of the antibody-mediated cytokine delivery approach, either as a single therapy or in combination with other agents.

The main aim of our work was to create a full-length bispecific antibody (BsAb) as a vehicle for the targeted delivery of interferon-beta (IFN-β) to ErbB2+ tumor cells in the form of non-covalent complex of BsAb and IFN-β. Such a construct is a CrossMab-type BsAb, consisting of an ErbB2-recognizing trastuzumab moiety, a part of chimeric antibody to IFN-β, and human IgG1 Fc domain carrying knob-into-hole amino acid substitutions necessary for the proper assembly of bispecific molecules. The IFN-β- recognizing arm of BsAb not only forms a complex with the cytokine but neutralizes its activity, thus providing a mechanism to avoid the side effects of the systemic action of IFN-β by blocking IFN-β Interaction with cell receptors in the process of cytokine delivery to tumor sites. Enzyme sandwich immunoassay confirmed the ability of BsAb to bind to human IFN-β comparable to that of the parental chimeric mAb. The BsAb binds to the recombinant ErbB2 receptor, as well as to lysates of ErbB2+ tumor cell lines. The inhibition of the antiproliferative effect of IFN-β by BsAb (IC50 = 49,3 µg/mL) was demonstrated on the HT29 cell line. It can be proposed that the BsAb obtained can serve as a component of the immunocytokine complex for the delivery of IFN-β to ErbB2-associated tumor cells.

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.

The development of and research into new therapies that can selectively and effectively destroy tumor cells that overexpress the ErbB2 receptor is apressing task. Recently, research into the use of type I interferons in the treatment of cancer has intensified. Cytokine therapy is aimed at activating the cells of the immune system to fight tumors, but it has drawbacks that limit its use because of a number of side effectsthe severity of which varies depending on the dosage and type of used cytokine. At the moment, a number of studies are being conducted regarding the use of IFNin oncology. The studies areaimed at mitigating the systemic action of this cytokine. The immunocytokine complex made of a bispecific antibody against the ErbB2 receptor and recombinant IFNdeveloped in this study underlies themechanism meant to avoid the systemic action of this cytokine. Part of this study focuses on the development of full-length antibodies that bind to the ErbB2 receptor on the one hand, and bind and neutralize IFN, on the other hand, which allows us to consider the antibodies as a means of cytokine delivery to tumor cells.

Humanization of antibodies for the development of novel therapeutic agents with low immunogenicity remains a topical problem in modern science. In the present work we describe the humanization of murine antibody B16 which binds and neutralizes human interferon-beta using the CDR-grafting method. Based on amino acid sequences of humanized and murine antibodies we constructed models of variable domains, analyzed, and compared them. The genes of humanized antibody hB16 and chimeric antibody chB16 were expressed in transient CHO cells. Antibodies were recovered from conditioned media, purified using affinity chromatography, and their properties were studied by biochemical and immunochemical methods. It was proven that humanized antibody hB16 possesses the same properties as murine mAb B16. This humanized antibody hB16 will be used in further work in order to obtain therapeutic immune complex composed of human interferon-beta and bispecific antibody which binds interferon-beta and the ErbB2 receptor.

— The extracellular part of the ErbB2 receptor (ecdErbB2), an oncological marker and a target for therapeutic antibodies, has been expressed in eukaryotic CHO cells. The extracellular part of the ErbB2 receptor has been isolated from a culture medium by metal affinity chromatography and characterized by biochemical and immunochemical methods. It has been shown that the receptor is produced in monomeric, dimeric, and trimeric forms. The oligomeric forms have been separated by gel filtration, and their ability to bind to Herceptin, one of the main drugs in the treatment of breast cancer, has been characterized by ELISA. The receptor produced can be used for in vitro immunochemical experiments.

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.

We determined the nucleotide and amino acid sequences of variable domains of three new monoclonal antibodies to the glycoprotein of Ebola virus capsid. The framework and hypervariable regions of immunoglobulin heavy and light chains were identified. The primary structures were confirmed using massspectrometry analysis. Immunoglobulin database search showed the uniqueness of the sequences obtained.

Balb/С mice were immunized with recombinant Ebola virus glycoprotein. Following the selection, screening, and cloning of murine hybridomas, we obtained five genetically stable clones of monoclonal antibodies GPE118 (IgG), GPE274 (IgM), GPE325 (IgM), GPE463 (IgM), and GPE534 (IgG). These antibodies were isolated and purified from the ascitic fluid of Balb/С mice using Protein G affinity chromatography (for IgG) and euglobulin precipitation (for IgM). To select at least three candidate antibodies for testing in biological assays as components of an antibody cocktail for the prophylaxis and treatment of hemorrhagic fever, we carried out an immunochemical analysis of the epitope specificity of the isolated antibodies. Based on the data of immunoblotting and sandwich ELISA, it became evident that the epitope recognized by GPE 534 differs from the epitopes recognized by the monoclonal antibodies GPE 118 and GPE 325. The last two antibodies also have different epitope specificity: it follows from the immunoblotting data and from the data on the binding of these antibodies with the intact and oxidized (partly deglycosylated) recombinant glycoprotein. For the biological activity studies and the development of recombinant counterparts, we selected three candidate high-affinity monoclonal antibodies GPE 534, GPE 118, and GPE 325.

BALB/с mice were immunized with recombinant Ebola virus glycoprotein. Following selection, screening and cloning of murine hybridomas we obtained 5 genetically stable clones of monoclonal antibodies GPE118 (IgG), GPE274 (IgM), GPE325 (IgM), GPE463 (IgM), and GPE534 (IgG). These antibodies were isolated and purified from ascitic fluid of BALB/с mice using Protein G affinity chromatography (IgG) and euglobulin precipitation method (IgM). For the selection of at least 3 candidate antibodies to be tested in biological assays as components of an antibody cocktail for the prophylaxis and treatment of hemorrhagic fever, we carried out an immunochemical analysis of epitope specificity of isolated antibodies. Based on immunoblotting and sandwich ELISA data, it became evident that the epitope recognized by GPE 534 is different from GPE 118 and GPE 325 epitopes. The latter two antibodies also have different epitope specificity. It is evidenced from immunoblotting data as well as from binding data of these antibodies with intact and oxidized (partly deglycosylated) recombinant glycoprotein. For the studies of biological activity and the development of recombinant counterparts, we isolated 3 candidate high-affinity monoclonal antibodies GPE 534, GPE 118, and GPE 325.

Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

The cell line CHO DG44 producing recombinant antibodies(Abs) to human tumor necrosis factor-alpha has been obtained. The influence of cell inoculation density and cultivation protocols on the level of Ab biosynthesis has been studied. The highest Ab yields have been observed at the inoculation density 3×106 cells/ml. The alternative method to cells-in-suspension cultivation has been proposed, which is the cell cultivation in calcium alginate hydrogel microgranules or alginate chitosan semipermeable microcapsules. It has been shown that the Ab production level by CHO DG44 cells entrapped into polymer microcapsules exceeds that of the cells-in-suspension cultivation regime.