ABSTRACT
Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM). However, reduced productivity and growth rate have been linked to miR-21 overexpression in CHO cells. The current study aimed to engineer a recombinant CHO (rCHO) cell using the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system coupled with the Bxb1 recombinase-mediated cassette exchange (RMCE) to express a circular miR-21 decoy (CM21D) with five bulged binding sites for miR-21 sponging. Implementing the ribonucleoprotein (RNP) delivery method, a landing pad was inserted into the genome utilizing the CRIS-PITCh technique. Subsequently, the CM21D cassette flanked by Bxb1 attB was then retargeted into the integrated landing pad using the RMCE/Bxb1 system. This strategy raised the targeting efficiency by 1.7-fold, and off-target effects were decreased. The miR-21 target genes (Pdcd4 and Atp11b) noticed a significant increase in expression upon the miR-21 sponging through CM21D. Following the expression of CM21D, rCHO cells showed a substantial decrease in doubling time and a 1.3-fold increase in growth rate. Further analysis showed an increased yield of hrsACE2, a secretory recombinant protein, by 2.06-fold. Hence, we can conclude that sponging-induced inhibition of miR-21 may lead to a growth rate increase that could be linked to increased CHO cell productivity. For industrial cell lines, including CHO cells, an increase in productivity is crucial. The results of our research indicate that CM21D is an auspicious CHO engineering approach. KEY POINTS: ⢠CHO is an ideal host cell line for producing industrial therapeutics manufacturing, and miR-21 is downregulated in CHO cells, which produce recombinant proteins. ⢠The miR-21 target genes noticed a significant increase in expression upon the miR-21 sponging through CM21D. Additionally, sponging of miR-21 by CM21D enhanced the growth rate of CHO cells. ⢠Productivity and growth rate were increased in CHO cells expressing recombinant hrs-ACE2 protein after CM21D knocking in.
Subject(s)
CRISPR-Cas Systems , Cricetulus , MicroRNAs , CHO Cells , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Cell Engineering/methods , Gene Editing/methods , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinases/genetics , Recombinases/metabolism , CricetinaeABSTRACT
Antibody drug conjugates (ADCs) with twelve FDA approved drugs, known as a novel category of anti-neoplastic treatment created to merge the monoclonal antibody specificity with cytotoxicity effect of chemotherapy. However, despite many undeniable advantages, ADCs face certain problems, including insufficient internalization after binding, complex structures and large size of full antibodies especially in targeting of solid tumors. Camelid single domain antibody fragments (Nanobody®) offer solutions to this challenge by providing nanoscale size, high solubility and excellent stability, recombinant expression in bacteria, in vivo enhanced tissue penetration, and conjugation advantages. Here, an anti-human CD22 Nanobody was expressed in E.coli cells and conjugated to Mertansine (DM1) as a cytotoxic payload. The anti-CD22 Nanobody was expressed and purified by Ni-NTA resin. DM1 conjugated anti-CD22 Nanobody was generated by conjugation of SMCC-DM1 to Nanobody lysine groups. The conjugates were characterized using SDS-PAGE and Capillary electrophoresis (CE-SDS), RP-HPLC, and MALDI-TOF mass spectrometry. Additionally, flow cytometry analysis and a competition ELISA were carried out for binding evaluation. Finally, cytotoxicity of conjugates on Raji and Jurkat cell lines was assessed. The drug-to-antibody ratio (DAR) of conjugates was calculated 2.04 using UV spectrometry. SDS-PAGE, CE-SDS, HPLC, and mass spectrometry confirmed conjugation of DM1 to the Nanobody. The obtained results showed the anti-CD22 Nanobody cytotoxicity was enhanced almost 80% by conjugation with DM1. The binding of conjugates was similar to the non-conjugated anti-CD22 Nanobody in flow cytometry experiments. Concludingly, this study successfully suggest that the DM1 conjugated anti-CD22 Nanobody can be used as a novel tumor specific drug delivery system.
Subject(s)
Immunoconjugates , Maytansine , Neoplasms , Single-Domain Antibodies , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/immunology , Cell Line, Tumor , Immunoconjugates/chemistry , Immunoconjugates/therapeutic use , Maytansine/chemistry , Neoplasms/drug therapy , Sialic Acid Binding Ig-like Lectin 2/immunology , Camelidae/immunologyABSTRACT
BACKGROUND: Conventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hot spots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell line development depends on an acceptable integration rate and robust sites for the sustained expression. METHODS AND RESULTS: In this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line. CONCLUSION: Our study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell line development.
Subject(s)
CRISPR-Cas Systems , Chromosomes, Human, Pair 3 , Animals , Cricetinae , Humans , CRISPR-Cas Systems/genetics , CHO Cells , Clone Cells , Cell Differentiation , CricetulusABSTRACT
CRISPR-mediated integration could be used to develop the recombinant CHO (rCHO) cells by knock-in into the hotspot loci. However, low HDR efficiency besides the complex donor design is the main barrier for achieving so. The recently introduced MMEJ-mediated CRISPR system (CRIS-PITCh) uses a donor with short homology arms, being linearized in the cells via two sgRNAs. In this paper, a new approach to improve CRIS-PITCh knock-in efficiency by employing small molecules was investigated. Two small molecules, B02, a Rad51 inhibitor, and Nocodazole, a G2/M cell cycle synchronizer, were used to target the S100A hotspot site using a bxb1 recombinase comprised landing pad in CHO-K1 cells. Following transfection, the CHO-K1 cells were treated with the optimum concentration of one or combination of small molecules, being determined by the cell viability or flow cytometric cell cycle assay. Stable cell lines were generated and the single-cell clones were achieved by the clonal selection procedure. The finding showed that B02 improved the PITCh-mediated integration approximately twofold. In the case of Nocodazole treatment, the improvement was even more significant, up to 2.4-fold. However, the combinatorial effects of both molecules were not substantial. Moreover, according to the copy number and out-out PCR analyses, 5 and 6 of 20 clonal cells exhibited mono-allelic integration in Nocodazole and B02 groups, respectively. The results of the present study as the first attempt to enhance the CHO platform generation by exploiting two small molecules in the CRIS-PITCh system could be used in future researches to establish rCHO clones.
Subject(s)
CRISPR-Cas Systems , DNA Repair , Cricetinae , Animals , Nocodazole , CHO Cells , CricetulusABSTRACT
For industrial production of recombinant protein biopharmaceuticals, Chinese hamster ovary (CHO) cells represent the most widely adopted host cell system, owing to their capacity to produce high-quality biologics with human-like posttranslational modifications. As opposed to random integration, targeted genome editing in genomic safe harbor sites has offered CHO cell line engineering a new perspective, ensuring production consistency in long-term culture and high biotherapeutic expression levels. Corresponding the remarkable advancements in knowledge of CRISPR-Cas systems, the use of CRISPR-Cas technology along with the donor design strategies has been pushed into increasing novel scenarios in cell line engineering, allowing scientists to modify mammalian genomes such as CHO cell line quickly, readily, and efficiently. Depending on the strategies and production requirements, the gene of interest can also be incorporated at single or multiple loci. This review will give a gist of all the most fundamental recent advancements in CHO cell line development, such as different cell line engineering approaches along with donor design strategies for targeted integration of the desired construct into genomic hot spots, which could ultimately lead to the fast-track product development process with consistent, improved product yield and quality.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Cricetinae , Animals , Humans , Cricetulus , CHO Cells , Recombinant Proteins/geneticsABSTRACT
Recombinant Chinese hamster ovary (CHO) cell line development for complex biotherapeutic production is conventionally based on the random integration (RI) approach. Due to the lack of control over the integration site and copy number, RI-generated cell pools are always coupled with rigorous screening to find clones that satisfy requirements for production titers, quality, and stability. Targeted integration into a well-defined genomic site has been suggested as a possible strategy to mitigate the drawbacks associated with RI. In this work, we employed the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system in combination with the Bxb1 recombinase-mediated cassette exchange (RMCE) system to generate an isogenic transgene-expressing cell line. We successfully utilized the CRIS-PITCh system to target a 2.6 kb Bxb1 landing pad with homology arms as short as 30 bp into the upstream region of the S100A gene cluster, achieving a targeting efficiency of 10.4%. The platform cell line (PCL) with a single copy of the landing pad was then employed for the Bxb1-mediated landing pad exchange with an EGFP encoding cassette to prove its functionality. Finally, to accomplish the main goal of our cell line development method, the PCL was applied for the expression of a secretory glycoprotein, human recombinant soluble angiotensin-converting enzyme 2 (hrsACE2). Taken together, on-target, single-copy, and stable expression of the transgene over long-term cultivation demonstrated our CRIS-PITCh/RMCE hybrid approach might possibly improve the cell line development process in terms of timeline, specificity, and stability. KEY POINTS: ⢠CRIS-PITCh system is an efficient method for single copy targeted integration of the landing pad and generation of platform cell line ⢠Upstream region of the S100A gene cluster of CHO-K1 is retargetable by recombinase-mediated cassette exchange (RMCE) approach and provides a stable expression of the transgene ⢠CRIS-PITCh/Bxb1 RMCE hybrid system has the potential to overcome some limitations of the random integration approach and accelerate the cell line development timeline.
Subject(s)
Genome , Recombinases , Cricetinae , Animals , Humans , CHO Cells , Cricetulus , Recombinases/genetics , TransgenesABSTRACT
BACKGROUND: Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli). METHODS: Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum. RESULTS: Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (Ë1.6-fold), and binding activity (Ë2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability. CONCLUSION: We were able to produce 21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies.
Subject(s)
Leukemia, Myeloid , Single-Chain Antibodies , Humans , Enzyme-Linked Immunosorbent Assay , Escherichia coli/metabolism , Interleukin-1 Receptor Accessory Protein/metabolism , Single-Chain Antibodies/metabolism , Inclusion BodiesABSTRACT
Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.
Subject(s)
Tissue Plasminogen Activator , Process Optimization , Flow Cytometry/methods , Fluorescence , Cell Count/instrumentation , Clone Cells/classification , Plasminogen Activator Inhibitor 1/adverse effects , Green Fluorescent ProteinsABSTRACT
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+).
Subject(s)
Antibodies, Bacterial/analysis , Antigens, Bacterial/immunology , Bacterial Toxins/immunology , Pore Forming Cytotoxic Proteins/immunology , Pseudomonas aeruginosa , Single-Chain Antibodies/analysis , Temperature , Time FactorsABSTRACT
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.
Subject(s)
Antigens, CD19/biosynthesis , Antigens, CD19/genetics , Hepatitis B Virus, Woodchuck/genetics , Hepatitis B Virus, Woodchuck/metabolism , Transcription, Genetic/physiology , Animals , CHO Cells , Cricetinae , Cricetulus , Gene Expression , HEK293 Cells , Humans , Jurkat CellsABSTRACT
Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and ï¬uorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.
Subject(s)
CRISPR-Cas Systems , Animals , CHO Cells , CRISPR-Cas Systems/genetics , Cricetinae , Cricetulus , Recombinant Proteins/genetics , TransgenesABSTRACT
OBJECTIVES: Immunotherapy using recombinant monoclonal antibodies specifically Anti-amyloid-beta (Anti-Aß) scFv is envisaged as an appropriate therapeutic for Alzheimer through reduction of amyloid-beta aggregation. The solubilization of therapeutics using polymeric micelles facilitates an improved bioavailability and extended blood half-life. In this study, the optimum production condition for Anti-amyloid-beta (Anti-Aß) scFv was obtained. To increase the stability of plasma, Anti-Aß-loaded polymeric micelles were synthesized. METHODS: Escherichia coli SHuffle expression strain was used and purified by Ni-NTA. Pluronics P85 and F127 micelles were used for the Anti-Aß delivery and were characterized in terms of morphology, drug loading and drug release in phosphate buffer and artificial cerebrospinal fluid. The stability profile was quantified at 4°C over a 30 days storage period. The stability in human plasma was also evaluated. KEY FINDINGS: Proteins expressed in SHuffle resulted in increased levels of protein expression and solubility. Low critical micelle concentration value and high micelle encapsulation efficiency (<200 nm) achieved via direct dissolution method. Anti-Aß-loaded micelles were around 2.2-fold more stable than Anti-Aß in plasma solution. A sustained in-vitro release of Anti-Aß from micelles was observed. CONCLUSIONS: Results confirmed that Pluronic-micelles pose benefits as a nano-carrier to increase the stability of Anti-Aß scFvin in the plasma.
Subject(s)
Amyloid beta-Peptides/immunology , Antibodies, Monoclonal/pharmacology , Biological Availability , Immunotherapy/methods , Solubility , Alzheimer Disease/drug therapy , Drug Carriers/pharmacology , Humans , Immunologic Factors/pharmacology , Micelles , Nanostructures , Poloxamer/pharmacology , Polymers/pharmacology , Recombinant Proteins/pharmacology , Surface-Active Agents/pharmacologyABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Toxins/antagonists & inhibitors , Cross Infection/drug therapy , Pore Forming Cytotoxic Proteins/antagonists & inhibitors , Pseudomonas Infections/drug therapy , Single-Chain Antibodies/pharmacology , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/therapeutic use , Antigens, Bacterial/metabolism , Bacterial Toxins/metabolism , Cell Line, Tumor , Cloning, Molecular , Computer Simulation , Cross Infection/immunology , Cross Infection/microbiology , Half-Life , Humans , Molecular Docking Simulation , Pore Forming Cytotoxic Proteins/metabolism , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/immunology , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Single-Chain Antibodies/genetics , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/therapeutic useABSTRACT
This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein scaffold to the Epithelial Cell Adhesion Molecule (EpCAM) Cancer Stem Cell (CSC) marker and its efficiency in targeted delivery of small interfering RNA (siRNA) molecules into the studied cells. Gene fragment encoding the DARPIn Ec1-LMWP fusion protein was subcloned into pET28a expression vector following molecular docking studies performed to examine the affinity of the fusion protein towards EpCAM marker. The binding of the siRNA to the expressed fusion protein was tested through native PAGE. The toxicity of the fusion protein was tested by MTT assay. Attachment of the complex to the EpCAM marker was investigated by flow cytometry and delivery of siRNA into the cells was assessed by fluorescence microscopy. The expressed 21.6 kDa DARPin Ec1-LMWP fusion protein was purified and showed no cytotoxicity on tested cells. Arginine rich LMWP was efficiently bounded to the negatively charged siRNA molecule. Successful attachment of the fusion protein:siRNA complex to the EpCAM marker and its internalization into MCF-7 breast cancer cell line were confirmed. Here for the first time the recombinant DARPin Ec1-LMWP protein scaffold was designed and tested for targeting EpCAM surface marker and successful internalization of the siRNA into MCF-7 cells.
Subject(s)
Biomarkers, Tumor/metabolism , Drug Carriers , Epithelial Cell Adhesion Molecule/metabolism , RNA, Small Interfering , Recombinant Fusion Proteins , Drug Carriers/chemistry , Drug Carriers/pharmacology , HeLa Cells , Humans , MCF-7 Cells , RNA, Small Interfering/chemistry , RNA, Small Interfering/pharmacology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacologyABSTRACT
Background: It is believed that the loading value of anticancer drug conjugated to the monoclonal antibody, called drug-to-antibody ratio (DAR), is the main quality feature of antibody-drug conjugates. Methods: In this study, matrix assisted laser desorption/ionization mass spectrometry was used to determine the average molecular weight of trastuzumab and its three conjugated forms. The differences in the measured masses for each conjugate and unconjugated trastuzumab were compared to the expected mass change through the conjugation of one mole of related drug-linker, in order to measure DAR. Results: There was a consistency between the loading results of mass spectrometry and the measurements of UV spectrometry in most cases. Conclusion: According to our findings, the MALDI-MS method for determining the loading values can be used rapidly and reliably to estimate the covalently bound drugs conjugated to antibodies when ESI-TOF-MS is unavailable.
Subject(s)
Antibodies, Monoclonal/analysis , Antineoplastic Agents/analysis , Immunoconjugates/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Molecular Weight , Peptides/analysis , Reproducibility of Results , Spectrophotometry, Ultraviolet , Trastuzumab/analysis , Trastuzumab/chemistry , Trypsin/metabolismABSTRACT
In recent decades, immunotoxins have attracted significant attention in treatment of a wide range of diseases including cancers due to their natural origins and their role in blocking crucial pathways within the cells. Ribosome inactivating proteins (RIPs) are efficient molecules in blocking protein synthesis through interactions with ribosomal rRNA molecules. cDNA molecule encoding HER2 scFv antibody fragment originated from trastuzumab attached to the mature alpha luffin gene fragment was subcloned into pET28a expression vector and expressed in different E. coli expression hosts. Identity of the expressed recombinant protein was investigated through western blotting and the fusion protein was purified using Ni-NTA affinity chromatography. The biological activity (toxicity) of the protein was investigated on DNA and RNA samples. A 58 kDa protein was expressed in E. coli. The best protein expression level was achieved in 0.2 mM IPTG at 30 °C in TB medium using E. coli BL21 (DE3) host strain. The fusion protein showed RNase and DNA glycosylase activity on tested RNA and DNA samples. DNA glycosylase activity of the recombinant fusion protein showed that alpha luffin part of this protein is active in conjugation to the scFv molecule and the expressed protein can be further studied in targeted biological in vitro assays.
Subject(s)
Cloning, Molecular/methods , Escherichia coli/genetics , Immunotoxins/genetics , Ribosome Inactivating Proteins, Type 1/genetics , Single-Chain Antibodies/genetics , Trastuzumab/genetics , Cell Line , Genetic Vectors/genetics , Humans , Immunotoxins/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Ribosome Inactivating Proteins, Type 1/pharmacology , Single-Chain Antibodies/pharmacology , Trastuzumab/pharmacologyABSTRACT
The crucial role of VEGF receptor 2 (VEGFR2) signaling in the angiogenesis and metastasis of solid tumors has prompted the development of inhibitors with minimal bystander effects. Recently, Adnectin C has attracted attention for cancer treatment. To overcome the problematic properties of Adnectin, a novel form of Adnectin C has been designed by its fusion to a biodegradable polymeric peptide containing Pro/Ala/Ser (PAS) repetitive residues. E. coli-expressed recombinant fused and unfused proteins were compared in terms of bioactivity, physicochemical, and pharmacokinetic properties using standard methods. Dynamic light scattering (DLS) analysis of PASylated adnectin C revealed an approximate 2-fold increase in particle size with a slight change in the net charge. Additionally, fusion of the PAS sequence improved its stability against the growth of thermo-induced aggregated forms. The high receptor-binding and improved binding kinetic parameters of PASylated Adnectin C was confirmed by ELISA and surface plasmon resonance assays, respectively. Pharmacokinetic studies showed a noticeable increase in the terminal half-life of Adnectin C-PAS#1(200) by a factor of 4.57 after single dose by intravenous injection into female BALB/c mice. The results suggest that PASylation could offer a superior delivery strategy for developing Adnectin-derived drugs with improved patient compliance.
Subject(s)
Fibronectins/pharmacology , Fibronectins/pharmacokinetics , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Alanine , Animals , Escherichia coli , Female , Fibronectins/isolation & purification , Fibronectins/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Kinetics , Mice , Mice, Inbred BALB C , Peptide Fragments/pharmacokinetics , Peptide Fragments/pharmacology , Proline , Protein Domains/physiology , Protein Engineering/methods , Serine , Surface Plasmon Resonance/methods , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Antibody-drug conjugates are now of considerable interest and are recommended for the treatment of cancers. Linkers are having a crucial role in potency and efficacy of these drugs. Herein, for the first time, we have used a water-soluble poly-ethylene glycol based linker (succinimidyl-[(N-maleimido propionamido)-diethyleneglycol] [SM(PEG)2]) for lysine amide coupling of DM1 drug to trastuzumab considering evaluation of the effect of using a hydrophilic linker on physicochemical and biological properties of the resulting conjugate in comparison to the conjugate containing succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker, which has a relative hydrophobic nature. The physicochemical properties of synthesized conjugates were investigated in terms of drug to antibody ratio, size variants and free drug quantities. In vitro biological activity of trastuzumab-DM1 conjugates was assessed on breast cancer cell lines expressing different levels of HER2 using binding affinity, antiproliferative, apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Synthesized conjugate containing hydrophilic linker, showed higher drug to antibody ratio, no aggregated form and higher cellular toxicity in comparison to SMCC bearing conjugate. Binding affinity and ADCC potential of conjugates was not affected upon the usage of hydrophilic linker. In conclusion, application of SM(PEG)2 for coupling of DM1 to trastuzumab enhance desirable characteristics of the resulting conjugate.
Subject(s)
Immunoconjugates/chemistry , Trastuzumab/chemistry , Trastuzumab/pharmacology , Antibodies/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Humans , Immunoconjugates/pharmacology , MCF-7 Cells , Maleimides/chemistry , Receptor, ErbB-2/metabolismABSTRACT
Aggregation of recombinant proteins, a major problem in E. coli expression system, is improved by using EnBase culture system based on slow release of glucose. In the present study, to understand the intracellular mechanisms involved in increased solubility of the target recombinant protein through EnBase system, the effect of this system was investigated on E. coli cells proteome profile. The proteome profile of E. coli cells cultured in EnBase and conventional batch mode was analyzed by two-dimensional gel electrophoresis. The proteins with significant expressional changes were identified through MALDI-TOF/TOF mass spectrometry. In EnBase system, the expressions of carbon metabolism-related proteins, sugar transport system-related proteins, and amino acids metabolism-related proteins were significantly altered. Furthermore, the expression of Thioredoxin 1 as the facilitator of protein folding was up-regulated in EnBase system that could be related to the increased solubility of recombinant protein. The proteomics analysis of E. coli cells cultured in EnBase system revealed that Thioredoxin 1 can be a potential candidate for future studies aiming at increased anti-VEGF fab fragment solubility. Studying proteomics is a valuable tool for revealing the target proteins that play the central role in EnBase culture system for increasing the solubility.
Subject(s)
Escherichia coli/genetics , Immunoglobulin Fab Fragments/genetics , Proteomics/methods , Vascular Endothelial Growth Factor A/immunology , Electrophoresis, Gel, Two-Dimensional , Immunoglobulin Fab Fragments/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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.