Construction of a Mammalian IRES-based Expression Vector to Amplify a Bispecific Antibody; Blinatumomab.

Blinatumomab, the bispecific T cell engager antibody (BsAb), has been demonstrated as the most successful BsAb to date. Throughout the past decade, vector design has great importance for the expression of monoclonal antibody in Chinese hamster ovary (CHO) cells. It has been indicated that expression vectors based on the elongation factor-1 alpha (EF-1 alpha) gene and DHFR selection marker can be highly effective to produce populations of stably transfected cells in the selection medium. Moreover, the phiC31 integrase system is considered as an attractive and safe protein expression system in mammalian cells and it could integrate a donor plasmid of any size, as a single copy, in to the host genome with no cofactors. In this study, phiC31 integrase technology in combination with DHFR amplification system was used to have an expression vector for future expression of blinatumomab in CHO cells. The gene of interest (BsAb gene) could be joined to DHFR selection marker with the insertion of an internal ribosome entry site (IRES). By positioning the DHFR downstream of BsAb gene and IRES, the transcription of the selection marker can depend on the successful transcription of the BsAb gene, which was located upstream in the expression construct. In this study, FC550A-1 vector was used as the backbone and DHFR selection marker was successfully combined with phiC31 integrase technology to generate a high-expressing construct for BsAb expression in CHO-DG44 cells in future studies.

The Epigenetic Regulation of Blinatumomab Gene Expression: Tumor Cell-dependent T cell Response against Lymphoma Cells and Cytotoxic Activity.

Conventional treatment for cancer such as surgical resection and chemotherapy can cause damage in cases with advanced cancers. Moreover, the identification of tumor-specific targets has great importance in T-cell therapies. For decades, T cell activity has been stimulated to improve anti-tumor activity. Bispecific antibodies have attracted strong interest from pharmaceutical companies, for their diagnostic and therapeutic use. Blinatumomab is a first-in-class bispecific T engager antibody for the treatment of relapsed or refractory precursor B- cell acute lymphoblastic leukemia. But, it can benefit several cases with CD19+ malignancies in the future. PhiC31 integrase-based vectors could selectively integrate therapeutic transgenes into pseudo-attP sites in CHO genome. In this study, production of Blinatumomab in CHO cells using this type of vectors was investigated. We evaluated the effects of histone deacetylases (HDACs) inhibitors such as sodium butyrate and valproic acid, on specific productivity and cell viability of antibody expressing cells. Although sodium butyrate increased specific productivity about 1.7-fold and valproic acid about 1.4-fold, valproic acid was found more efficient because of its less cytotoxic effect on cell growth. We examined the efficacy of expressed Blinatumomab at various effector to target (E/T) ratios. A dose-response analyses of calcein-acetoxymethyl release assay illustrated that the effective dose of expressed mAb required for antibody mediated cytotoxicity was 100 ng/ml and the expressed mAb was more effective at E/T ratios of 10:1 and 5:1. Results of this study indicated that the expressed blinatumomab can be useful for enhancing the cytotoxicity of CD3+ T-cells against CD19 + target cells in vitro.

A comparative study of the bispecific monoclonal antibody, blinatumomab expression in CHO cells and E. coli.

The "bispecifics" market improved over the past decade due to the development of many technological platforms including bispecific T cell engagers (BiTEs). The approval of blinatumomab, the most advanced bispecific T-cell engager (BiTE) in clinical trials, can be a significant milestone in the development of bispecific antibodies. Both Chinese hamster ovary (CHO) cells and E. coli strain are considered as the most widely used hosts for the large-scale production of therapeutic monoclonal antibodies. Since both of the economic and qualitative aspects of protein production are important in industry, selection of a suitable protein expression system is very critical. The BsAb gene was cloned into the expression vectors FC550A-1, pcDNA3.1 (+), and PET22b and 6 × His-tagged BsAb then purified on a Ni-NTA chromatography column. Both SDS-PAGE and Western blotting analysis of the purified protein demonstrated that blinatumomab was successfully expressed as a 55 kDa in both expression systems. The antigen-binding properties of blinatumomab were compared in the mammalian system versus Escherichia coli. The results showed that the purified antibody from a mammalian expression system has better binding activity than the one from E. coli host.

Anti-CD19 Monoclonal Antibodies: a New Approach to Lymphoma Therapy.

CD19 is expressed on B- lineage cells and follicular dendritic cells and plays a key role in B cell malignancies and autoimmune diseases. Thus, it has been considered as potential target for several monoclonal antibodies (mAbs). For decades, chemotherapy has been known as one of the major antitumor therapies eradicating high proliferative tumor cells. But, anti- CD19 mAbs developed for treating CD19- positive lymphomas and autoimmune diseases would rank among the most novel area of research and development in the pharmaceutical industry. Moreover, several anti- CD19 mAbs are currently being tested in various clinical trials and this review provides an overview of the research accomplished so far.

Effects of imatinib mesylate in mouse models of multiple sclerosis and in vitro determinants.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS), This autoimmune disease is mainly mediated by adaptive and innate immune responses that lead to an inflammatory demyelination and axonal damage. Imatinib mesylate is a selective protein tyrosine kinase inhibitor with immunomodulatory properties that abrogates multiple signal transduction pathways in immune cells. In the present research, our aim was to test the therapeutic efficacy of imatinib in experimental model of MS. We performed EAE induction in 23 female C57 mice by myelin oligodendrocyte glycoprotein-35-55 (MOG35-55) in Complete Freund's Adjuvant (CFA) emulsion and used imatinib for treatment of EAE. The clinical evaluation and histopathology were assessed. Also for in vitro analysis, we used U-87 MG, C6 and WEHI-164 cell lines to evaluate the inhibitory effects of imatinib in cell proliferation, as well as pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6) and matrix metalloproteinase (MMP) secretion. Our findings demonstrated that this drug had beneficial effects on EAE by attenuation in the severity and a delay in the onset of disease. In vitro, imatinib inhibited cell proliferation, MMP-2 expression and activity and also attenuated the production of proinflammatory cytokines. Imatinib with its potential therapeutic effects and immunomodulatory properties may be considered, after additional necessary tests and trials, for treatment of MS.

Novel therapeutic approach by nicotine in experimental model of multiple sclerosis.

BACKGROUND: Multiple sclerosis is an autoimmune, neurodegenerative disease of the central nervous system. The cause of multiple sclerosis is still unknown, and there is no cure for multiple sclerosis. Experimental autoimmune encephalomyelitis is considered as an animal model for multiple sclerosis. The therapeutic role of nicotine has been proven to be effective in both Alzheimer's and Parkinson's disease, thus we examined, for the first time, the role of nicotine in the experimental autoimmune encephalomyelitis model. METHODS: Experimental autoimmune encephalomyelitis induction was performed according to Guang-Xian Zhang et al. Treatment with nicotine was started on Day 7 post-immunization. Prevention with nicotine was started on Day 7 pre-immunization. Also for in-vitro analysis, we used U-87 MG cell line to evaluate the inhibitory effect of nicotine in cell proliferation, pro-inflammatory cytokines (TNF-alpha, IL-lbeta, IL-6) and MMP-2 activity by MTT, ELISA, and zymoanalysis methods, respectively. Moreover, the brains of mice were removed for histological analysis. RESULTS: Our findings showed that treatment with nicotine caused a significant reduction in the severity and onset of the experimental autoimmune encephalomyelitis. Histological analysis indicated that there was very mild and mild plaque in the brain sections of nicotine prevention and treatment groups, respectively. CONCLUSION: Our data indicate that nicotine can significantly improve the clinical score and attenuate the demyelinating pathology typically found in experimental autoimmune encephalomyelitis, indicating that nicotine has protective effects in experimental model of multiple sclerosis.

The neglected role of histamine in Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by beta-amyloid plaques accumulation and cognitive impairment. Both environmental factors and heritable predisposition have a role in AD. Histamine is a biogenic monoamine that plays a role in several physiological functions, including induction of inflammatory reactions, wound healing, and regeneration. The Histamine mediates its functions via its 4 G-protein-coupled Histamine H1 receptor (H1R) to histamine H1 receptor (H4R). The histaminergic system has a role in the treatment of brain disorders by the development of histamine receptor agonists, antagonists. The H1R and H4R are responsible for allergic inflammation. But recent studies show that histamine antagonists against H3R and regulation of H2R can be more efficient in AD therapy. In this review, we focus on the role of histamine and its receptors in the treatment of AD, and we hope that histamine could be an effective therapeutic factor in the treatment of AD.

Natural killer cells and their role in rheumatoid arthritis: friend or foe?

Rheumatoid arthritis (RA) is a long-term disease that leads to inflammation of the joints and surrounding tissues. Natural killer (NK) cells are an important part of the innate immune system and are responsible for the first line of defense against pathogens during the initial immune challenge before the adaptive immune system eventually eliminates the infectious burden. NK cells have the capacity to damage normal cells or through interaction with other cells such as dendritic cells, macrophages, and T cells cause autoimmune diseases, such as RA. NK cells isolated from the joints of patients with RA suggest that they may play a role in this disease. However, the involvement of NK cells in RA pathology is not fully elucidated. Both protective and detrimental roles of NK cells in RA have recently been reported. A better understanding of NK cells' role in RA might help to develop new therapeutic strategies for treatment of the RA or other autoimmune diseases. We have decided in this paper to focus on the NK cell biology, and attempt to bring the interested readership of this Journal up to date on the NK cell, specifically its possible relation to RA.