Development of Stable CHO-K1 Cell Lines Overexpressing Full-Length Human CD20 Antigen.

Background: CD20 is a differentiation-related antigen exclusively expressed on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders, making it an ideal target for immunotherapy of hematological malignancies and autoimmune diseases. MAb-based therapies targeting CD20 have a principal role in the treatment of several immune-related disordes and cancers, including CLL. Fc gamma receptors mediate CD20 internalization in hematopoietic cells; therefore, this study aimed to establish non-hematopoietic stable cell lines overexpressing full-length human CD20 antigen as an in vitro model for CD20-related studies. Methods: CD20 gene was cloned into the transfer vector. The lentivirus system was transfected to packaging HEK 293T cells, and the supernatants were harvested. CHO-K1 cells were transduced using recombinant viruses, and a stable cell pool was developed by the antibiotic selection. CD20 expression was confirmed at the mRNA and protein levels. Results: Simultaneous expression of GFP protein facilitated the detection of CD20-expressing cells. Immunophenotyping analysis of stable clones demonstrated expression of CD20 antigen. In addition, the mean fluorescence intensity was significantly higher in the CD20-CHO-K1 clones than the wild-type CHO-K1 cells. Conclusion: This study is the first report on using second-generation lentiviral vectors for the establishment of a non-hematopoietic cell-based system, which stably expresses full-length human CD20 antigen. Results of stable CHO cell lines with different levels of CD20 antigen are well suited to be used for CD20-based investigations, including binding and functional assays.

Development of an Expression Vector Engineering Strategy Based on tDNA Insulator Element for the Stable Expression of Vascular Endothelial Growth Factor Receptor-Fc Fusion Protein.

During the past decades, tremendous advances have occurred in manufacturing recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. Nevertheless, the production of stable high-producing cell lines has remained a major obstacle in the development process of the CHO cell line. It has been shown that genomic regulatory elements can promote cell line development efficiency by improving transgenes' productivity and stability. Such elements include insulators, ubiquitous chromatin opening elements, scaffold/matrix attachment regions, and antirepressors. In addition, tDNA elements are shown to act as insulators in mammalian cells. This study examines the effect of the tDNA insulator on stable expression of a vascular endothelial growth factor receptor-Fc fusion protein.

Association of serum leptin with angiographically proven cardiovascular disease and with components of the metabolic syndrome: a cross-sectional study in East Azerbaijan.

BACKGROUND: Role of leptin is well documented in cardiometabolic diseases. The objective of this study was to investigate if the serum levels of leptin associates with the serum levels of markers related to cardiac and metabolic disorders in adults. MATERIALS AND METHODS: One hundred eighty subjects [120 cardiovascular disease (CVD) and 60 healthy controls] were enrolled in the study, to determine the association of the serum leptin (in quartiles) and cardiometabolic diseases [metabolic syndrome (MetS) and CVD] adjusted for other biological and physical examination. MetS was according to the WHO Clinical Criteria for MetS definition and CVD by angiography outcomes. The serum levels of leptin and OX-LDL were measured by ELISA. RESULTS: Leptin levels were significantly higher in patients with MetS and those with positive angiography compared with controls. After controlling for potential confounders, a significant association of the leptin levels with cardiometabolic diseases was proven, albeit there was a higher rate of significance between CVD and leptin in comparison with MetS. Additionally, receiver operating characteristic analysis revealed that the serum levels of leptin were a valuable biomarker of the cardiometabolic diseases. CONCLUSION: Our findings demonstrate that serum leptin levels are associated with components of the MetS and with CVD. Serum leptin may be a useful biomarker for CVD.

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris.

BACKGROUND: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. RESULTS: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. CONCLUSIONS: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

Improving the expression of anti-IL-2Rα monoclonal antibody in the CHO cells through optimization of the expression vector and translation efficiency.

The growing need for monoclonal antibodies (mAbs) necessitates the development of novel and efficient production approaches. Regulatory elements like ubiquitous chromatin-opening elements (UCOEs) have been employed for improvement of the mAb expression in the Chinese hamster ovary (CHO) cells. SINEUPs are a class of long non-coding RNAs, which can improve the translation of partly overlapping mRNAs. A combination of these two elements might lead to higher production of mAbs. Therefore, the current study was conducted to investigate the effects of SINEUPs and A2UCOE on the expression of an IgG1 in the CHO-K1 cells. Hence, after constructing the mAb, mAb-SINEUP, and mAb-UCOE vectors, four stable cell pools were generated through combining the above vectors. According to the expression analysis, antibody yields were higher in the mAb-SINEUP and mAb-UCOE cell pools compared to the mAb cells. In addition, the cells possessing both SINEUP and UCOE elements provided the best expression. Persistent mAb expression was observed for over 2 months in these cells, whilst the expression was decreased in the mAb pool. SINEUP and UCOE positively influenced the stable mAb expression. It can be concluded that the SINEUP and UCOE enhance the antibody stability and expression level separately and their combination improves the mAb production in the CHO cells.

The cooperative effects of micro-grooved topography and TGF-ß1 on the vascular smooth muscle cell contractile protein expression of the mesenchymal stem cells.

Cell morphological changes induced by micro-grooved topography have been shown to be an important regulator of smooth muscle (SM) differentiation of mesenchymal stem cells (MSCs). In addition to the micro-grooved topography, transforming growth factor-ß1 (TGF-ß1) can also modulate MSCs differentiation towards smooth muscle cells (SMCs) through alterations in cell morphological characteristics. Thus, it can be hypothesized that substrate topography and TGF-ß1 may interact to facilitate differentiation of MSCs into SMCs. In this study, we investigated the time-course cooperative effects of substrate topography and TGF-ß1 in the regulation of SM differentiation of human MSCs. Western blotting, followed by image analysis, was performed to assess the protein expression of α-actin, h1-calponin and gelsolin. Three-way analysis of variance was employed to investigate the main effect of each independent variable, i.e. TGF-ß1 conditioning, substrate topography and culture time, along with the interactions of these variables. Each of TGF-ß1, substrate topography and culture time significantly affected the protein expression of α-actin, h1-calponin and gelsolin. Overall, TGF-ß1 conditioning of the cells and culturing the cells on the micro-grooved substrate resulted in greater protein expression of α-actin and h1-calponin, and lesser protein expression of gelsolin. In addition to the isolated effects of the variables, treatment type interacted with substrate topography and culture time to regulate the expression of the above-mentioned proteins. This study indicated the feasibility of promoting SM differentiation of human MSCs by simultaneous recruitment of micro-grooved topography and TGF-ß1. The findings could be of assistance when effective utilization of chemo-physical cues is needed to achieve functional SMC-like MSCs in vitro.

Yeast Expression Systems: Overview and Recent Advances.

Yeasts are outstanding hosts for the production of functional recombinant proteins with industrial or medical applications. Great attention has been emerged on yeast due to the inherent advantages and new developments in this host cell. For the production of each specific product, the most appropriate expression system should be identified and optimized both on the genetic and fermentation levels, considering the features of the host, vector and expression strategies. Currently, several new systems are commercially available; some of them are private and need licensing. The potential for secretory expression of heterologous proteins in yeast proposed this system as a candidate for the production of complex eukaryotic proteins. The common yeast expression hosts used for recombinant proteins' expression include Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, Arxula adeninivorans, Kluyveromyces lactis, and Schizosaccharomyces pombe. This review is dedicated to discuss on significant characteristics of the most common methylotrophic and non-methylotrophic yeast expression systems with an emphasis on their advantages and new developments.

The applications of anti-CD20 antibodies to treat various B cells disorders.

B-lymphocyte antigen CD20 (called CD20) is known as an activated-glycosylated phosphoprotein which is expressed on the surface of all B-cells. CD20 is involved in the regulation of trans-membrane Ca2+ conductance and also play critical roles in cell-cycle progression during human B cell proliferation and activation. The appearance of monoclonal antibody (mAb) technology provided an effective field for targeted therapy in treatment of a variety of diseases such as cancer, and autoimmune diseases. Anti-CD20 is one of important antibodies which could be employed in treatment of several diseases. Increasing evidences revealed that efficacy of different anti-CD20 antibodies is implicated by their function. Hence, evaluation of anti-CD20 antibodies function could provide and introduce new anti-CD20 based therapies. In the present study, we summarized several applications of anti-CD20 antibodies in various immune related disorders including B-CLL (B-cell chronic lymphocytic leukemia), rheumatoid arthritis (RA), multiple sclerosis (MS) and melanoma.

Functional mutations in and characterization of VHH against Helicobacter pylori urease.

Manipulation of clinically significant antibodies can effectively improve the processes of diagnosis and treatment. Affinity maturation process has a significant role in improvement of antibodies efficiency. Error-prone PCR technique is one of the proposed methods for improvement of the affinity of antibodies. In the present research, a method was applied to camel heavy-chain antibody (VHH, nanobody) raised against UreC subunit of urease enzyme from Helicobacter pylori. This VHH was used as a starting molecule to construct a highly diversified phage displayed VHH library. The constructed library of nanobody mutants was subjected to several rounds of panning against UreC antigen. High-affinity mutant was selected. Our VHH (HMR23) showed 1.5-fold higher binding activity than the parental VHH. In addition, the mutant VHH presented a better performance in inhibition of urease activity at low concentrations retaining its specificity and thermal stability.