Expression, Purification, and Biological Evaluation of XTEN-GCSF in a Neutropenic Rat Model.

Granulocyte colony-stimulating factor (GCSF) stimulates the proliferation of neutrophils but it has low serum half-life. Therefore, the present study was done to investigate the effect of XTENylation on biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a neutropenic rat model. XTEN tag was genetically fused to the N-terminal region of GCSF-encoding gene fragment and subcloned into pET28a expression vector. The cytoplasmic expressed recombinant protein was characterized through intrinsic fluorescence spectroscopy (IFS), dynamic light scattering (DLS), and size exclusion chromatography (SEC). In vitro biological activity of the XTEN-GCSF protein was evaluated on NFS60 cell line. Hematopoietic properties and pharmacokinetics were also investigated in a neutropenic rat model. An approximately 140 kDa recombinant protein was detected on SDS-PAGE. Dynamic light scattering and size exclusion chromatography confirmed the increase in hydrodynamic diameter of GCSF molecule after XTENylation. GCSF derivatives showed efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF represented the lowest EC50 value (100.6 pg/ml). Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer could significantly increase protein serum half-life in comparison with the commercially available GCSF molecules. PEGylated and XTENylated GCSF proteins were more effective in stimulation of neutrophils compared to the GCSF molecule alone. XTENylation of GCSF represented promising results in in vitro and in vivo studies. This approach can be a potential alternative to PEGylation strategies for increasing serum half-life of protein.

Recombinant anti-human epidermal growth factor receptor type 2 single-chain variable fragment-alpha-luffin fusion protein as a putative immunotoxin against human epidermal growth factor receptor type 2-positive breast cancer cells: an experimental research.

Background: Breast cancer is one of the most frequent causes of cancer death in women. The application of immunotoxins to target overexpressed biomarkers on the surface of cancer cells and delivery of the toxin molecules into these cells has attracted too much attention during the last decade. Objectives: This study was conducted to investigate the possible in-vitro cytotoxic and apoptotic activity of previously designed recombinant immunotoxin compromising anti-HER2 single-chain variable fragment (scFv) and alpha-luffin protein in human epidermal growth factor receptor type 2 (HER2)-positive and HER2-negative breast cancer cell lines. Materials and methods: The previously designed recombinant immunotoxin and alpha-luffin protein were expressed in E. coli host cells and purified using Ni-affinity chromatography. The cytotoxicity of the proteins was tested through MTT and apoptosis studies on HER2-positive and HER2-negative breast cancer cell lines. Results: Treatment of SKBR3 and MDA-MB-468 cells with the immunotoxin caused differential cytotoxicity and apoptotic events. Flow cytometry analysis indicated that the immunotoxin could arrest SKBR3 cells at the G0/G1 phase and induce apoptosis and cell death which were not observed in HER2-negative MDA-MB-468 cells. Annexin V/PI staining revealed late apoptotic events in SKBR3 cells treated with the immunotoxin which was different from the early apoptosis induced by the alpha-luffin protein alone. Conclusions: This immunotoxin could be a promising tool in developing new targeted therapeutic agents against HER2-positive cancer cells. Animal experiments are needed before making firmed conclusions.

Extension of human GCSF serum half-life by the fusion of albumin binding domain.

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Computational analysis of fusion protein of anti-HER2 scFv and alpha luffin: A new immunotoxin protein for HER2 positive cancers

Abstract The present study deals with the computational design and analysis of a novel fusion protein based on a single chain variable fragment that binds to the extracellular domain of human epidermal growth factor receptor 2 (HER2) in breast cancer cells. Alpha luffin, a small ribosome inactivating protein (RIP), was attached to the anti-HER2 antibody fragment. I-TASSER modeling provided the full-length structure of the fusion protein. Molecular docking evaluated the molecular interactions of the complementarity-determining regions of designed fusion protein to HER2. Energy minimization and molecular dynamics simulations were conducted to refine the complexes. RMSD plot revealed reasonable stability of the fusion protein during the simulation. The free binding energy profile of complexes affirmed a favorable binding affinity of proteins in complex with HER2 using molecular mechanics Poisson-Boltzmann surface area (G-MMPBSA) algorithm. In general, this approach looks promising in the development of new fusion proteins in terms of immunotoxins with appropriate cytotoxicity.

Woodchuck Hepatitis Virus Post-Transcriptional Regulation Element (WPRE) Promotes Anti-CD19 BiTE Expression in Expi293 Cells.

Background: Bispecific antibodies represent an important class of monoclonal antibodies (mAbs), with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti-CD3 × CD19 bispecific antibody (bsAb) is a bispecific T-cell engager currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts. Methods: Woodchuck hepatitis virus post-transcriptional regulation (WPRE) sequence was incorporated at the 3' end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion: Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in human embryonic kidney-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody.

Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule.

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

Optimization of culture conditions for high-level expression of soluble and active tumor necrosis factor-α in E. coli.

Anti-TNF inhibitors exert their therapeutic effect by inhibition of the excessive amounts of TNF-α within the body. Recombinant TNF-α should be produced in a soluble refolded form to investigate the effectiveness and efficiency of anti-TNF-α compounds. In this research, the designed cassette was subcloned in the pET28a expression vector and expressed in E. coli BL21 (DE3). The identity of the protein was confirmed through SDS-PAGE and Western blotting. After optimizing expression conditions, protein purification was performed using native Ni-NTA affinity chromatography. The biological activity of the soluble recombinant TNF-α was investigated using MTT assay. Also, the affinity of an anti-TNF-α agent, Altebrel, was investigated against the expressed protein through ELISA. Optimization of TNF-α expression conditions represented that the highest expression could be achieved at 37 °C using 0.5 mM IPTG 6 h post-induction. The recombinant protein represented an inhibitory effect on the L929 murine fibroblast cell line and was successfully detected by Altebrel in ELISA. Binding kinetics were also studied using Cimzia as an anti-TNF-α molecule and 7.2 E-13M was calculated as the equilibrium dissociation constant value (KD). The significant expression level of the recombinant protein in the soluble form, its high purity, and assessment of its biological activity showed that the expressed protein could be used in tests of ELISA and MTT to assess the activity of anti-TNF-α agents.

Improvement of Certolizumab Fab' properties by PASylation technology.

Certolizumab pegol is a Fab' antibody fragment for treatment of rheumatoid arthritis and Crohn's disease which is conjugated to a 40 kDa PEG molecule in order to increase the protein half-life. PEGylation may have disadvantages including immunogenicity, hypersensitivity, vacuolation, decreased binding affinity and biological activity of the protein. To overcome these problems, PASylation has been developed as a new approach. The nucleotide sequence encoding 400 amino acid PAS residues was genetically fused to the corresponding nucleotide sequences of both chains of certolizumab. Then, the bioactivity as well as physicochemical and pharmacokinetic properties of the recombinant PASylated expressed protein was assayed. Circular dichroism spectroscopy demonstrated that the random coil structure of PAS sequences did not change the secondary structure of the PASylated Fab' molecule. It was observed that PASylation influenced the properties of the Fab' molecule by which the hydrodynamic radius and neutralization activity were increased. Also, the antigen binding and binding kinetic parameters improved in comparison to the PEGylated Fab' antibody. Pharmacokinetic studies also showed prolonged terminal half-life and improved pharmacokinetic parameters in PASylated recombinant protein in comparison to the PEGylated and Fab' control molecules. The results reconfirmed the efficiency of PASylation approach as a potential alternative method in increasing the half-life of pharmaceutical proteins.

Comparison of Linear Poly Ethylene Imine (LPEI) and Poly L-Lysine (PLL) in Fabrication of CHOK1 Cell-Loaded Multilayer Alginate Microcapsules.

Purpose: Poly l-lysine (PLL) has been introduced as a strengthening covering layer for alginate microcapsules which are the most convenient way for cell encapsulation. Some disadvantages of PLL such as high price and low biocompatibility have prompted scientists to find better alternatives. Linear poly ethylene imine (LPEI), thanks to its highly similar structure to PLL, could be considered as a proper cost-effective alternative. In this study LPEI and PLL were compared as covering layers of cell-loaded alginate-LPEI-alginate (cALA) and alginate-PLL-alginate (cAPA) microcapsules. Methods: In addition to the physico-mechanical properties, the encapsulation efficiency, cell survival post encapsulation, cell viability, and cellular metabolic activity within the microcapsules were evaluated using trypan blue, live/dead cell staining, and MTT test, respectively. Results: Physico-mechanical evaluation of the microcapsules revealed that the cell microencapsulation process did not affect their shape, size, and mechanical stability. Although the encapsulation efficiency for cALA and cAPA was not different (P >0.05), cell survival post encapsulation was higher in cALA than in cAPA (P<0.05) which could be the reason for the higher cell viability and also cellular metabolic activity within these microcapsules in comparison to cAPA. Conclusion: Here, based on these results, ALA could be introduced as a preferable alternative to APA for cell encapsulation.

Anti-HER2 scFv Expression in Escherichia coli SHuffle®T7 Express Cells: Effects on Solubility and Biological Activity.

Breast cancer is the second most commonly diagnosed cancer, worldwide. Human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer is correlated with poor prognosis. HER2-targeting monoclonal antibodies resulted in longer survival of HER2+ breast cancer. Single-chain variable fragment (scFv) demonstrates improved penetrability into tumors. Due to the presence of two disulfide bond, scFv expression in reducing bacterial cytoplasm may cause formation of inclusion bodies. Disulfide bond can be formed properly in cytoplasm of SHuffle® strain as it is trxB-, gor-, and overexpresses cytoplasmic DsbC chaperone. In this study, the anti-HER2 scFv was successfully expressed and purified in BL21 (DE3) and SHuffle® cells. Here, significant higher soluble anti-HER2 scFv was produced in SHuffle® than in BL21 strain. The specific binding of anti-HER2 scFv to HER2 was shown by flow cytometry analysis and ELISA. Moreover, it was demonstrated that the anti-HER2 scFv produced in SHuffle® binds to HER2 at higher level as compared to that expressed in BL21 cells. Furthermore, competitive ELISA-based study suggested that anti-HER2 scFv recognizes the same epitope of HER2 receptor as the trastuzumab antibody. Our findings indicated that correct disulfide bond formation in SHuffle® strain can result in enhanced solubility and higher biological activity level of anti-HER2 scFv.

The improvement of anti-HER2 scFv soluble expression in Escherichia coli

The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2+ tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemicallysynthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 ºC. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 ºC).

Effect of Cysteamine on Cell Growth and IgG4 Production in Recombinant Sp2.0 Cells.

The manipulation of redox potential in secretory pathway by thiol reducing agents can be a strategy to improve the production levels of disulfide-bonded proteins including recombinant antibodies. Here we have studied the influence of cysteamine on viability and the production level of IgG4 in Sp2.0 cells. For this purpose, the recombinant Sp2.0 cells producing an anti CD33 IgG4, were subjected to different concentrations of cysteamine. At concentrations of 2, 4 and 5 mM cysteamine, the secreted levels of IgG4 did not change significantly. However, in concentration of 7 mM cysteamine, a significant decrease was observed in IgG4 levels which may indicate the cytotoxicity of this compound in higher concentrations. Our results show that the cysteamine treatment reduces the cell viability in a dose-dependent manner. Also it was observed that 2 mM cysteamine had no late effect on IgG4 production level and only at day 3, this concentration of cysteamine decreased the cell viability significantly. To test whether the addition of cysteamine can affect the expression level of protein disulfide isomerase, RT-PCR analysis was carried out. The results revealed that cysteamine does not affect the PDI transcription and expression level of IgG4 in this type of recombinant cells.