RÉSUMÉ
OBJECTIVE: The anticancer properties of recombinant α-luffin (LUF) are wellestablished. However, the cytotoxic effects of encapsulating LUF within niosomes on the SKBR3 breast cancer cell line have yet to be explored. Our study aimed to investigate whether this encapsulation strategy could improve cytotoxic effects. METHODS: Alpha-luffin was expressed, purified, and refolded. Then, this protein was utilized to craft an optimal formulation, guided by experimental design. In this work, we have explored various physicochemical properties, including particle size, polydispersity index, zeta potential, morphology, entrapment efficiency, drug release and kinetics, storage stability, and FTIR spectroscopy. Additionally, we have assessed the cellular uptake and cytotoxic effect of the optimized niosome formulation on the SKBR3 breast cancer cell line. RESULTS: The optimized niosome exhibited a mean diameter of 315±6.4 nm (DLS). Successful encapsulation of LUF into regularly shaped, spherical niosomes was achieved, with an encapsulation efficiency of 73.45±2.4%. Notably, Niosomal LUF (NLUF) exhibited significantly increased cytotoxicity against SKBR3 cells. CONCLUSION: These findings suggest that niosomes loaded with LUF hold promise as a potential treatment strategy for breast cancer.
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The baculovirus expression vector system (BEVS) is a powerful tool in pharmaceutical biotechnology to infect insect cells and produce the recombinant proteins of interest. It has been well documented that optimizing the culture condition and its supplementation through designed experiments is critical for maximum protein production. In this study, besides physicochemical parameters including incubation temperature, cell count of infection, multiplicity of infection, and feeding percentage, potential supplementary factors such as cholesterol, polyamine, galactose, pluronic-F68, glucose, L-glutamine, and ZnSO4 were screened for Spodoptera frugiperda (Sf9) cell culture and expression of hemagglutinin (HA) protein of Influenza virus via Placket-Burman design and then optimized through Box-Behnken approach. The optimized conditions were then applied for scale-up culture and the expressed r-HA protein was characterized. Optimization of selected parameters via the Box-Behnken approach indicated that feed percentage, cell count, and multiplicity of infection are the main parameters affecting r-HA expression level and potency compared to the previously established culture condition. This study demonstrated the effectiveness of designing experiments to select and optimize important parameters that potentially affect Sf9 cell culture, r-HA expression, and its potency in the BEVS system.
Sujet(s)
Glycoprotéine hémagglutinine du virus influenza , Spodoptera , Animaux , Cellules Sf9 , Glycoprotéine hémagglutinine du virus influenza/métabolisme , Glycoprotéine hémagglutinine du virus influenza/génétique , Techniques de culture cellulaire/méthodes , Milieux de culture , Protéines recombinantes/génétique , Protéines recombinantes/métabolisme , Protéines recombinantes/biosynthèse , Baculoviridae/génétique , Baculoviridae/métabolismeRÉSUMÉ
The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.
Sujet(s)
Acétaminophène , Anticorps monoclonaux , Survie cellulaire , Anticorps monoclonaux/pharmacologie , Animaux , Acétaminophène/pharmacologie , Survie cellulaire/effets des médicaments et des substances chimiques , Souris , Protéines du choc thermique HSP70/métabolisme , Protéines du choc thermique HSP70/génétique , Protéines du choc thermique/génétique , Protéines du choc thermique/métabolisme , Bioréacteurs , Réaction de choc thermique/effets des médicaments et des substances chimiques , Protéines du choc thermique HSP27/métabolisme , Protéines du choc thermique HSP27/génétique , Lignée cellulaireRÉSUMÉ
BACKGROUND: Interleukin 2 (IL-2) is a vital cytokine in the induction of T and NK cell responses, the proliferation of CD8+ T cells, and the effective treatment of human cancers such as melanoma and renal cell carcinoma. However, widespread use of this cytokine is limited due to its short half-life, severe toxicity, lack of specific tumor targeting, and activation of Treg cells mediated by high-affinity interleukin-2 receptors. OBJECTIVE: In this study, a tumor-targeting LIV-1 VHH-mutIL2 immunocytokine with reduced CD25 (α chain of the high-affinity IL-2 receptor) binding activity was developed to improve IL-2 half-life by decreasing its renal infiltration in comparison with wild and mutant IL-2 molecules. METHODS: The recombinant immunocytokine was designed and expressed. The biological activity of the purified fusion protein was investigated in in vitro and in vivo experiments. RESULTS: The fusion protein represented specific binding to MCF7 (the breast cancer cell line) and more efficient cytotoxicity than wild-type IL-2 and mutant IL-2. The PK parameters of the recombinant immunocytokine were also improved in comparison to the IL-2 molecules. CONCLUSION: The observed results showed that LIV1-mIL2 immunocytokine could be considered as an effective agent in the LIV-1-targeted treatment of cancers due to its longer half-life and stronger cytotoxicity.
Sujet(s)
Antinéoplasiques , Interleukine-2 , Humains , Interleukine-2/métabolisme , Interleukine-2/immunologie , Animaux , Antinéoplasiques/pharmacologie , Antinéoplasiques/composition chimique , Souris , Femelle , Protéines tumorales/immunologie , Protéines tumorales/métabolisme , Protéines tumorales/génétique , Prolifération cellulaire/effets des médicaments et des substances chimiques , Tests de criblage d'agents antitumoraux , Cellules MCF-7 , Anticorps à domaine unique/immunologie , Anticorps à domaine unique/pharmacologie , Souris de lignée BALB C , Protéines de fusion recombinantes/pharmacologie , Protéines de fusion recombinantes/génétique , Protéines adaptatrices de la transduction du signal , Protéines IAPRÉSUMÉ
The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.
RÉSUMÉ
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.
Sujet(s)
Facteur de stimulation des colonies de granulocytes , Polymères , Animaux , Rats , Facteur de stimulation des colonies de granulocytes/génétique , Facteur de stimulation des colonies de granulocytes/isolement et purification , Facteur de stimulation des colonies de granulocytes/métabolisme , Facteur de stimulation des colonies de granulocytes/pharmacologie , Granulocytes neutrophiles , Polymères/composition chimique , Protéines recombinantes/isolement et purification , Protéines recombinantes/métabolisme , Protéines recombinantes/pharmacologieRÉSUMÉ
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.
RÉSUMÉ
Objectives: One of the important interactions in controlling the human immune system is the reaction between checkpoint proteins such as programmed cell death-1 (PD-1) and its ligand, PD-L1. These are negative immunoregulatory molecules that promote immune evasion of tumor cells. PD-L1 expression is an immune-mediated mechanism used by various malignant cells in order to down-regulate the immune system. Checkpoint inhibitors (CPIs) are a new class of anti-cancer agents that stimulate immune cells to elicit an antitumor response by blocking the ligand and receptor interactions. Nanobody (Nb) as a new type of antibody fragment, has some potential as CPI. Materials and Methods: A female camel was immunized with recombinant PD-L1 protein, nanobody library was constructed and PD-L1 specific Nb was selected. The selected Nb was characterized in terms of affinity, specificity, and binding potency in ELISA, Western blotting, and flow cytometry. Results: Developed nanobody, A22 binds to its cognate target with high specificity and affinity. Western blot and flow cytometry techniques showed that nanobody A22 was able to specifically detect and attach to human PD-L1 protein on the cell surface and in the cell lysate. MTT assay showed the inhibitory effect of PD-L1 by specific Nb on A431 and HEK293 cells, with no cytotoxic effect on cell growth. Conclusion: The results highlighted the potential of anti-PD-L1 Nb as a novel therapeutic in cancer therapy without undesirable cytotoxicity.
RÉSUMÉ
Interleukin-2 (IL-2) is an important cytokine in survival, expansion, function of CD8+ T cells and natural killer cells in immunotherapy of melanoma and renal cell carcinomas. Its severe toxicity following binding to its high affinity IL-2 receptor alpha (IL-2Rα) has restricted its application in cancer patients. In the present study, we investigated the antitumor efficacy and cytotoxicity of a mutated human IL-2 previously designed by selective amino acid substitutions, and its reduced affinity towards high-affinity IL-2Rα (CD25) was approved compared to the wild type IL-2 (wtIL-2). Furthermore, their ability to induce PBMC cell proliferation, and interferon-gamma secretion was compared. The mutant IL-2 also represented higher antitumor activity and more efficient cytotoxicity than wild type hIL-2. The developed mutant IL-2 can be an alternative tool in IL-2 associated immunotherapy of various cancers.
Sujet(s)
Interleukine-2 , Mélanome , Humains , Interleukine-2/génétique , Interleukine-2/métabolisme , Interleukine-2/pharmacologie , Cellules tueuses naturelles/métabolisme , Agranulocytes/métabolisme , Mélanome/métabolisme , Récepteurs à l'interleukine-2/métabolismeRÉSUMÉ
Although high-dose IL-2 has clear antitumor effects, severe side effects like severe toxicity and activation of Tregs by binding of IL-2 to high-affinity IL-2R, hypotension, and vascular leak syndrome limit its applications as a therapeutic antitumor agent. Here in this study, a rational computational approach was employed to develop and design novel triple-mutant IL-2 variants with the aim of improving IL-2-based immunotherapy. The affinity of the mutants towards IL-2Rα was further computed with the aid of molecular dynamic simulations and umbrella sampling techniques and the obtained results were compared to those of wild-type IL-2. In vitro experiments by flow cytometry showed that the anti-CD25 mAb was able to bind to PBMC cells even after mutant 2 preincubation, however, the binding strength of the mutant to α-subunit was less than of wtIL-2. Additionally, reduction of IL-2Rα subunit affinity did not significantly disturb IL-2/IL2Rßγc subunits interactions.
Sujet(s)
Sous-unité alpha du récepteur à l'interleukine-2 , Agranulocytes/métabolisme , Simulation de docking moléculaire , Simulation de dynamique moléculaire , Électricité statique , Humains , Interleukine-2/composition chimique , Interleukine-2/métabolisme , Sous-unité alpha du récepteur à l'interleukine-2/composition chimique , Sous-unité alpha du récepteur à l'interleukine-2/métabolisme , Liaison aux protéinesRÉSUMÉ
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.
Sujet(s)
Albumines/pharmacologie , Prolifération cellulaire/effets des médicaments et des substances chimiques , Facteur de stimulation des colonies de granulocytes/sang , Facteur de stimulation des colonies de granulocytes/pharmacologie , Domaines protéiques , Albumines/composition chimique , Albumines/pharmacocinétique , Animaux , Lignée cellulaire , Phénomènes chimiques , Modèles animaux de maladie humaine , Escherichia coli , Facteur de stimulation des colonies de granulocytes/composition chimique , Facteur de stimulation des colonies de granulocytes/pharmacocinétique , Période , Humains , Corps d'inclusion , Numération des leucocytes , Neutropénie/métabolisme , Granulocytes neutrophiles/effets des médicaments et des substances chimiques , Liaison aux protéines , RatsRÉSUMÉ
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.
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Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results: : Increased efficiency was achieved by EnBase® compared to LuriaBertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 µg/mL. Conclusion: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).
Sujet(s)
Anticorps antibactériens/analyse , Antigènes bactériens/immunologie , Toxines bactériennes/immunologie , Perforines/immunologie , Pseudomonas aeruginosa , Anticorps à chaîne unique/analyse , Température , Facteurs tempsRÉSUMÉ
Background: Cystic echinococcosis is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods: The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results: Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. Mucosal IgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion: Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice.
Sujet(s)
Antigènes d'helminthe/immunologie , Echinococcus granulosus/immunologie , Protéines d'helminthes/immunologie , Immunité humorale/immunologie , Phénomènes immunogénétiques/physiologie , Lactococcus lactis/immunologie , Animaux , Antigènes d'helminthe/administration et posologie , Femelle , Protéines d'helminthes/administration et posologie , Immunité humorale/effets des médicaments et des substances chimiques , Phénomènes immunogénétiques/effets des médicaments et des substances chimiques , Souris , Souris de lignée BALB C , Protéines recombinantes/immunologieRÉSUMÉ
With the increasing dominance of monoclonal antibodies (mAbs) in the biopharmaceutical industry and smaller antibody fragments bringing notable advantages over full-length antibodies, it is of considerable significance to choose the most suitable production system. Although mammalian expression system has been the preferred choice in recent years for mAbs production, E. coli could be the favorable host for non-glycosylated small antibody fragments due to the emergence of new engineered E. coli strains capable of forming disulfide-bonds in their cytoplasm.In this study, non-glycosylated anti-TNF-α Fab' moiety of Certolizumab pegol, produced by periplasmic expression in E. coli in previous studies, was produced in the cytoplasm of E. coli SHuffle strain. The results indicated that it is biologically functional by testing the antigen-binding activity via indirect ELISA and inhibition of TNF-α induced cytotoxicity using MTT test. Major factors affecting protein production and, optimized culture conditions were examined by analyzing growth characteristics and patterns of expression in 24 h of post-induction cultivation and, optimization of culture conditions by response surface methodology considering temperature, time of induction and concentration of inducer in small (tube) and shake-flask scale. Based on the results, temperature had the most significant influence on functional protein yield while exerting different impacts in small and shake-flask scales, which indicated that cultivation volume is also an important factor that should be taken into account in optimization process. Furthermore, richness of medium and slower cellular growth rate improved specific cellular yield of functional protein by having a positive effect on the solubility of Fab' antibody.
Sujet(s)
Biomasse , Certolizumab pégol , Cytoplasme , Escherichia coli , Certolizumab pégol/biosynthèse , Certolizumab pégol/génétique , Cytoplasme/génétique , Cytoplasme/métabolisme , Escherichia coli/génétique , Escherichia coli/métabolisme , Humains , Protéines recombinantesRÉSUMÉ
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.
Sujet(s)
Antibactériens/pharmacologie , Toxines bactériennes/antagonistes et inhibiteurs , Infection croisée/traitement médicamenteux , Perforines/antagonistes et inhibiteurs , Infections à Pseudomonas/traitement médicamenteux , Anticorps à chaîne unique/pharmacologie , Antibactériens/isolement et purification , Antibactériens/usage thérapeutique , Antigènes bactériens/métabolisme , Toxines bactériennes/métabolisme , Lignée cellulaire tumorale , Clonage moléculaire , Simulation numérique , Infection croisée/immunologie , Infection croisée/microbiologie , Période , Humains , Simulation de docking moléculaire , Perforines/métabolisme , Infections à Pseudomonas/immunologie , Infections à Pseudomonas/microbiologie , Pseudomonas aeruginosa/effets des médicaments et des substances chimiques , Pseudomonas aeruginosa/immunologie , Protéines recombinantes/génétique , Protéines recombinantes/isolement et purification , Protéines recombinantes/pharmacologie , Protéines recombinantes/usage thérapeutique , Anticorps à chaîne unique/génétique , Anticorps à chaîne unique/isolement et purification , Anticorps à chaîne unique/usage thérapeutiqueRÉSUMÉ
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.
Sujet(s)
Escherichia coli/génétique , Protéines de fusion recombinantes , Facteur de nécrose tumorale alpha , Animaux , Lignée cellulaire , Chromatographie d'affinité , Milieux de culture/métabolisme , Humains , Souris , Repliement des protéines , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/isolement et purification , Protéines de fusion recombinantes/métabolisme , Facteur de nécrose tumorale alpha/composition chimique , Facteur de nécrose tumorale alpha/génétique , Facteur de nécrose tumorale alpha/isolement et purification , Facteur de nécrose tumorale alpha/métabolismeRÉSUMÉ
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.
Sujet(s)
Certolizumab pégol , Polyéthylène glycols , Animaux , Polyarthrite rhumatoïde/traitement médicamenteux , Lignée cellulaire , Certolizumab pégol/composition chimique , Certolizumab pégol/pharmacocinétique , Certolizumab pégol/pharmacologie , Maladie de Crohn/traitement médicamenteux , Femelle , Période , Humains , Souris , Souris de lignée BALB C , Polyéthylène glycols/composition chimique , Polyéthylène glycols/pharmacocinétique , Polyéthylène glycols/pharmacologieRÉSUMÉ
A number of mutations in the epidermal growth factor receptor (EGFR) have been identified that imparts resistance to anti-EGFR monoclonal antibodies (mAbs) in clinical and preclinical samples. Primary or acquired resistance to targeted therapy will eventually limit the clinical benefit of anticancer mAbs. The aim of the current study was to perform computational analysis to investigate the structural implications of the EGFR somatic mutations on its complexes with the four anti-EGFR mAbs (Cetuximab, Panitumumab, Necitumumab, and Matuzumab). Docking analysis and molecular dynamics (MD) simulations were performed to understand the plausible structural and dynamical implications caused by somatic mutations available in the Catalogue of Somatic Mutations in Cancer database on the EGFR and anti-EGFR mAbs. We found that EGFRS492R and EGFRV441I in complex with Cetuximab, EGFRR377S and EGFRS447Y in complex with Panitumumab, and EGFRV441I in complex with Necitumumab have a weakest binding affinity in comparison to EGFRWT in complex with the relevant mAb. Taken together with the results obtained from docking analysis and MD simulations, the present findings may suggest that, the S492R and V441I mutations confer resistance to Cetuximab, R377S and S447Y mutations mediate resistance to Panitumumab and finally, V441I mutation also confers resistance to Necitumumab.
Sujet(s)
Anticorps monoclonaux humanisés/pharmacologie , Antinéoplasiques immunologiques/pharmacologie , Cétuximab/pharmacologie , Panitumumab/pharmacologie , Anticorps monoclonaux humanisés/composition chimique , Cétuximab/composition chimique , Résistance aux médicaments antinéoplasiques , Récepteurs ErbB/composition chimique , Récepteurs ErbB/génétique , Humains , Mutation faux-sens , Tumeurs/traitement médicamenteux , Tumeurs/génétique , Panitumumab/composition chimique , Mutation ponctuelle , ThermodynamiqueRÉSUMÉ
Current advances in antibody engineering driving the strongest growth area in biotherapeutic agents development. Affinity improvement that is mainly important for biological activity and clinical efficacy of therapeutic antibodies, has still remained a challenging task. In the human body, during a course of immune response affinity maturation increase antibody activity by several rounds of somatic hypermutation and clonal selection in the germinal center. The final outputs are antibodies representing higher affinity and specificity against a particular antigen. In the realm of biotechnology, exploring of mutations which improve antibody affinity while preserving its specificity and stability is an extremely time-consuming and laborious process. Recent advances in computational algorithms and DNA sequencing technologies help researchers to redesign antibody structure to achieve desired properties such as improved binding affinity. In this review, we briefly described the principle of affinity maturation and different corresponding in vitro techniques. Also, we recapitulated the most recent advancements in the field of antibody affinity maturation including computational approaches and next-generation sequencing (NGS).