ABSTRACT
The TGF-ß family ligands myostatin, GDF11, and activins are negative regulators of skeletal muscle mass, which have been reported to primarily signal via the ActRIIB receptor on skeletal muscle and thereby induce muscle wasting described as cachexia. Use of a soluble ActRIIB-Fc "trap," to block myostatin pathway signaling in normal or cachectic mice leads to hypertrophy or prevention of muscle loss, perhaps suggesting that the ActRIIB receptor is primarily responsible for muscle growth regulation. Genetic evidence demonstrates however that both ActRIIB- and ActRIIA-deficient mice display a hypertrophic phenotype. Here, we describe the mode of action of bimagrumab (BYM338), as a human dual-specific anti-ActRIIA/ActRIIB antibody, at the molecular and cellular levels. As shown by X-ray analysis, bimagrumab binds to both ActRIIA and ActRIIB ligand binding domains in a competitive manner at the critical myostatin/activin binding site, hence preventing signal transduction through either ActRII. Myostatin and the activins are capable of binding to both ActRIIA and ActRIIB, with different affinities. However, blockade of either single receptor through the use of specific anti-ActRIIA or anti-ActRIIB antibodies achieves only a partial signaling blockade upon myostatin or activin A stimulation, and this leads to only a small increase in muscle mass. Complete neutralization and maximal anabolic response are achieved only by simultaneous blockade of both receptors. These findings demonstrate the importance of ActRIIA in addition to ActRIIB in mediating myostatin and activin signaling and highlight the need for blocking both receptors to achieve a strong functional benefit.
Subject(s)
Activin Receptors, Type II/antagonists & inhibitors , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Hypertrophy/chemically induced , Muscle, Skeletal/drug effects , Activin Receptors, Type II/metabolism , Activins/metabolism , Animals , Antibodies, Blocking/therapeutic use , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized , Bone Morphogenetic Proteins/metabolism , Crystallography, X-Ray , Dose-Response Relationship, Drug , Growth Differentiation Factors/metabolism , HEK293 Cells , Humans , Hypertrophy/pathology , Male , Mice , Mice, SCID , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Myostatin/metabolism , Rats , Rats, Wistar , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Wasting Syndrome/drug therapy , Wasting Syndrome/pathologyABSTRACT
Recombinant CHO (Chinese hamster ovary) cell lines producing therapeutic proteins often lose their production capability during long-term cultivation. To ensure that CHO production cell lines can be up-scaled to high-volume bioreactors, labor intensive stability studies of several months have to be performed to deselect clones that are losing productivity over time. The ability to predict whether clones will produce recombinant proteins at constant high levels, for example, through determination of biomarkers such as expression of specific genes, plasmid integration sites, or epigenetic patterns, or even to improve CHO host cell lines to increase the probability of the generation of stable clones would be highly beneficial. Previously, we reported that the lack of a telomeric region of chromosome 8 correlates with increased productivities and higher production stabilities of monoclonal antibody expressing CHO cell lines (Ritter A, Voedisch B, Wienberg J, Wilms B, Geisse S, Jostock T, Laux H. 2016a. Biotechnol Bioeng 113(5):1084-1093). Herein, we describe that the knock-out of the gene Fam60A, which is one of the genes located within the telomeric region of chromosome 8, in CHO-K1a cells leads to the isolation of significantly more clones with higher protein production stabilities of monoclonal antibodies during long-term cultivation. Biotechnol. Bioeng. 2017;114: 701-704. © 2016 Wiley Periodicals, Inc.
Subject(s)
Cell Engineering/methods , DNA-Binding Proteins/physiology , Recombinant Proteins/metabolism , Acetylation , Animals , Bioreactors , CHO Cells , Cricetinae , Cricetulus , DNA-Binding Proteins/genetics , Gene Knockout Techniques , Histones , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Recently, we reported that the loss of a telomeric region of chromosome 8 in Chinese Hamster Ovary (CHO) cells correlates with higher recombinant productivities. New cell lines lacking this region, called CHO-C8DEL, showed several advantages during cell line generation and for the production of recombinant proteins (Ritter et al., 2016, Biotechnol Bioeng). Here, we performed knock-down and knock-out experiments of genes located within this telomeric region of chromosome 8 to identify the genes causing the observed phenotypes of CHO-C8DEL cell lines. We present evidence that loss or reduced expression of the gene C12orf35 is responsible for higher productivities and shorter recovery times during selection pressure. These effects are mediated by increased levels of mRNA of the exogenes heavy chain (HC) and light chain (LC) as well as dihydrofolate reductase (DHFR) and neomycin phosphotransferase (Neo) during the stable expression of antibodies. Biotechnol. Bioeng. 2016;113: 2433-2442. © 2016 Wiley Periodicals, Inc.
Subject(s)
CHO Cells/physiology , Genetic Enhancement/methods , Recombinant Proteins/biosynthesis , Animals , CHO Cells/cytology , Cricetulus , Gene Knockdown Techniques , Recombinant Proteins/genetics , Up-Regulation/geneticsABSTRACT
Chinese Hamster Ovary (CHO) cells are widely used for large scale production of recombinant biopharmaceuticals. Although these cells have been extensively used, a demand to further increase the performance, for example, to facilitate the process of clone selection to isolate the highest producing cell lines that maintain stability of production over time is still existing. We compared gene expression profiles of high versus low producing CHO clones to identify regulated genes which can be used as biomarkers during clone selection or for cell line engineering. We present evidence that increased production rates and cell line stability are correlated with the loss of the telomeric region of the chromosome 8. A new parental CHO cell line lacking this region was generated and its capability for protein production was assessed. The average volumetric productivity of cells after gene transfer and selection was found to be several fold improved, facilitating the supply of early drug substance material to determine for example, quality. In addition, significantly more cell clones with a higher average productivity and higher protein production stability were obtained with the new host cell line after single cell cloning. This allows reduced efforts in single cell sorting, screening of fewer clones and raises the opportunity to circumvent time and labor-intensive stability studies.
Subject(s)
CHO Cells/metabolism , Chromosomes, Human, Pair 8/genetics , Telomere/genetics , Animals , Antibodies, Monoclonal/genetics , CHO Cells/cytology , Chromosome Deletion , Clone Cells/cytology , Clone Cells/metabolism , Cricetinae , Cricetulus , Down-Regulation , Gene Expression Regulation , Genetic Engineering/methods , Humans , Recombinant Proteins/genetics , Transcriptome , TransfectionABSTRACT
Protein production for structural and biophysical studies, functional assays, biomarkers, mechanistic studies in vitro and in vivo, but also for therapeutic applications in pharma, biotech and academia has evolved into a mature discipline in recent years. Due to the increased emphasis on biopharmaceuticals, the growing demand for proteins used for structural and biophysical studies, the impact of genomics technologies on the analysis of large sets of structurally diverse proteins, and the increasing complexity of disease targets, the interest in innovative approaches for the expression, purification and characterisation of recombinant proteins has steadily increased over the years. In this review, we summarise recent developments in the field of recombinant protein expression for research use in pharma, biotech and academia. We focus mostly on the latest developments for protein expression in the most widely used expression systems: Escherichia coli (E. coli), insect cell expression using the Baculovirus Expression Vector System (BEVS) and, finally, transient and stable expression of recombinant proteins in mammalian cells.
Subject(s)
Drug Discovery , Recombinant Proteins/biosynthesis , Baculoviridae , Cells, Cultured , Escherichia coli , Gene Expression , Genetic Vectors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Staining and Labeling , TransfectionABSTRACT
A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.
Subject(s)
Drug Design , Piperidines/chemistry , Piperidines/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Renin/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Inhibitory Concentration 50 , Models, Molecular , Piperidines/administration & dosage , Piperidines/pharmacokinetics , Protease Inhibitors/administration & dosage , Protease Inhibitors/pharmacokinetics , Protein Conformation , Rats , Renin/chemistryABSTRACT
Disease intervention by targeting a critical pathway molecule through a blocking antibody or interference by therapeutic proteins is currently en vogue. Generation of blocking antibodies or therapeutic proteins inevitably requires the production of recombinant proteins or cell-based immunogens. Thus, one could call the antigen molecule the neglected player in antibody drug discovery. The variety of methods available for making recombinant proteins or recombinant cell lines that present the target on the cell surface is extensive. These need to be addressed in conjunction with biochemical and biophysical quality criteria and the experimental application intended. Fundamentally, successful production and isolation of monoclonal antibodies requires optimized antigen preparation and presentation to the immune host. This review summarizes the most important aspects of antigen generation and display, enabling logical decision making to give rise to potent high-affinity antibodies.
Subject(s)
Antibodies/immunology , Antibodies/pharmacology , Antigens/biosynthesis , Antigens/immunology , Drug Discovery , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Animals , Cell Line , Cell Surface Display Techniques , Humans , ImmunizationABSTRACT
The first protocols describing transient gene expression in mammalian cells for the rapid generation of recombinant proteins emerged more than 10 years ago as an alternative to the establishment of stable, often amplified clonal cell lines, and relieved somewhat the bias against mammalian cell systems as being too complicated, labor intensive, and tedious to serve as a source for tool proteins in industrial research and academia. Over the past decade, these attempts have been refined and optimized, giving rise to expression protocols applicable in every lab in dependence on available tools, equipment, and envisaged outcome. This chapter summarizes the development of transient expression technologies over the past decade up to its current status and provides an outlook into what may be the future of transient technology development.
Subject(s)
CHO Cells/cytology , Genetic Techniques , HEK293 Cells/cytology , Recombinant Proteins , Transfection/methods , Animals , Cricetinae , Culture Media , Gene Expression , Genetic Vectors , Humans , Lipopeptides/chemistry , Lipopeptides/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
The impact of transient gene expression approaches (TGE) on the rapid production of recombinant proteins is undisputed, despite that all efforts are currently relying on two host cell families only, namely HEK293 derivatives and CHO cell line(s). Yet, the increasing complexity of biological targets calls for more than two host cell types to meet the challenges of difficult-to-express proteins. For this reason, we evaluated the more recently established novel CAP-T® cell line derived from human amniocytes for its performance and potential in transient gene expression. Upon careful analyses and adaptation of all process parameters we show here that indeed the CAP-T® cells are extremely amenable to transient gene expression and recombinant protein production. Additionally, they possess inherent capabilities to express and secrete complex and difficult target molecules, thus adding an attractive alternative to the repertoire of existing host cell lines used in transient production processes.
Subject(s)
Amniotic Fluid/cytology , Amniotic Fluid/metabolism , Recombinant Proteins/biosynthesis , Transfection/methods , Amniotic Fluid/chemistry , Blotting, Western , Cell Line , Gene Expression , Humans , Liposomes/chemistry , Plasmids/genetics , Polyethyleneimine/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/ß-catenin signaling. We found the extracellular ß-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.
Subject(s)
Bone Morphogenetic Proteins/metabolism , LDL-Receptor Related Proteins/metabolism , Osteocytes/metabolism , Osteogenesis , Adaptor Proteins, Signal Transducing , Amino Acid Substitution , Animals , Bone Morphogenetic Proteins/genetics , Genetic Markers/genetics , HEK293 Cells , Humans , LDL-Receptor Related Proteins/genetics , Mice , Mutation, Missense , Signal Transduction/genetics , Wnt1 Protein/genetics , Wnt1 Protein/metabolism , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
The timely availability of recombinant proteins in sufficient quantity and of validated quality is of utmost importance in driving drug discovery and the development of low molecular weight compounds, as well as for biotherapeutics. Transient gene expression (TGE) in mammalian cells has emerged as a promising technology for protein generation over the past decade as TGE meets all the prerequisites with respect to quantity and quality of the product as well as cost-effectiveness and speed of the process. Optimized protocols have been developed for both HEK293 and CHO cell lines which allow protein production at any desired scale up to >100 l and in milligram to gram quantities. Along with an overview on current scientific and technological knowledge, detailed protocols for expression of recombinant proteins on small, medium, and large scale are discussed in the following chapter.
Subject(s)
Gene Transfer Techniques , Mammals/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Eukaryotic Cells/chemistry , Eukaryotic Cells/metabolism , Humans , Mammals/metabolism , Recombinant Proteins/isolation & purificationABSTRACT
The Baculovirus Expression Vector System (BEVS) is one of the most efficient systems for production of recombinant proteins and consequently its application is wide-spread in industry as well as in academia. Since the early 1970s, when the first stable insect cell lines were established and the infectivity of bacu-lovirus in an in vitro culture system was demonstrated (1, 2), virtually thousands of reports have been published on the successful expression of proteins using this system as well as on method improvement. However, despite its popularity the system is labor intensive and time consuming. Moreover, adaptation of the system to multi-parallel (high-throughput) expression is much more difficult to achieve than with E. coli due to its far more complex nature. However, recent years have seen the development of strategies that have greatly enhanced the stream-lining and speed of baculovirus protein expression for increased throughput via use of automation and miniaturization. This chapter therefore tries to collate these developments in a series of protocols (which are modifications to standard procedure plus several new approaches) that will allow the user to expedite the speed and throughput of baculovirus-mediated protein expression and facilitate true multi-parallel, high-throughput protein expression profiling in insect cells. In addition we also provide a series of optimized protocols for small and large-scale transient insect cell expression that allow for both the rapid analysis of multiple constructs and the concomitant scale-up of those selected for on-going analysis. Since this approach is independent of viral propagation, the timelines for this approach are markedly shorter and offer a significant advantage over standard bacu-lovirus expression approach strategies in the context of HT applications.
Subject(s)
Baculoviridae/genetics , Insecta/genetics , Recombinant Proteins/biosynthesis , Animals , Baculoviridae/growth & development , Cell Culture Techniques , Cells/metabolism , Insecta/cytology , Plasmids , Transfection/methodsSubject(s)
Gene Expression , Transfection , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Humans , Transfection/methods , Transfection/trendsABSTRACT
We describe an efficient inducible gene expression system in HEK.EBNA cells, a well-established cell system for the rapid transient expression of research-tool proteins. The transgene control system of choice is the novel acetaldehyde-inducible regulation (AIR) technology, which has been shown to modulate transgene levels following exposure of cells to acetaldehyde. For application in HEK.EBNA cells, AlcR transactivator plasmids were constructed and co-expressed with the secreted alkaline phosphatase (SEAP) gene under the control of a chimeric mammalian promoter (P(AIR)) for acetaldehyde-regulated expression. Several highly inducible transactivator cell lines were established. Adjustable transgene induction by gaseous acetaldehyde led to high induction levels and tight repression in transient expression trials and in stably transfected HEK.EBNA cell lines. Thus, the AIR technology can be used for inducible expression of any desired recombinant protein in HEK.EBNA cells. A possible application for inducible gene expression is a controlled proliferation strategy. Clonal HEK.EBNA cell lines, expressing the fungal transactivator protein AlcR, were engineered for gas-adjustable expression of the cell-cycle regulator p27(Kip1). We show that expression of p27(Kip1) via transient or stable transfection led to a G1-phase specific growth arrest of HEK.EBNA cells. Furthermore, production pools engineered for gas-adjustable expression of p27(Kip1) and constitutive expression of SEAP showed enhanced productive capacity.
Subject(s)
Alkaline Phosphatase/genetics , G1 Phase/physiology , Gene Expression Regulation , Proliferating Cell Nuclear Antigen/metabolism , Recombinant Proteins/metabolism , Acetaldehyde/metabolism , Alkaline Phosphatase/metabolism , Bioreactors , Cell Line , Epstein-Barr Virus Nuclear Antigens/metabolism , Humans , Kidney/cytology , Kidney/embryology , Recombinant Proteins/biosynthesis , Transgenes/physiology , Viral Proteins/metabolismABSTRACT
Mammalian expression systems have an undisputed long-standing and very successful history for the generation of recombinant proteins, mainly as biopharmaceuticals. However, for use as 'tool proteins' in, e.g. assay development and screening, for structure elucidation and as antigens these expression systems were generally regarded as being cumbersome, tedious and expensive. This bias has largely been overcome with the very recent development of large-scale transient transfection (LST) approaches. Especially the HEK.EBNA expression system described here has contributed significantly to this success. The simplicity and speed of this approach compares well with expression trials using the widely applied Baculovirus/insect cell system. In addition, proteins generated in mammalian cells are usually correctly folded, fully processed and functionally active.
Subject(s)
Bioreactors , Epstein-Barr Virus Nuclear Antigens/genetics , Recombinant Proteins/biosynthesis , Transfection/methods , Cell Line , Culture Media , HumansSubject(s)
Gene Expression/genetics , Recombinant Proteins/genetics , Recombinant Proteins/therapeutic use , Transfection/methods , Animals , Calcium Phosphates , Cell Line , Humans , Mammals , Plasmids , Polyethyleneimine , Recombinant Proteins/metabolism , Time Factors , Transfection/instrumentation , Transformation, Genetic/geneticsABSTRACT
This report describes a method to culture insects cells in 24 deep-well blocks for the routine small-scale optimisation of baculovirus-mediated protein expression experiments. Miniaturisation of this process provides the necessary reduction in terms of resource allocation, reagents, and labour to allow extensive and rapid optimisation of expression conditions, with the concomitant reduction in lead-time before commencement of large-scale bioreactor experiments. This therefore greatly simplifies the optimisation process and allows the use of liquid handling robotics in much of the initial optimisation stages of the process, thereby greatly increasing the throughput of the laboratory. We present several examples of the use of deep-well block expression studies in the optimisation of therapeutically relevant protein targets. We also discuss how the enhanced throughput offered by this approach can be adapted to robotic handling systems and the implications this has on the capacity to conduct multi-parallel protein expression studies.
Subject(s)
Clinical Laboratory Techniques/methods , Animals , Baculoviridae , Cell Culture Techniques/methods , Cells, Cultured , Genetic Vectors , Kinetics , Phosphatidylinositol 3-Kinases/biosynthesis , Phosphatidylinositol 3-Kinases/genetics , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Receptor, Adenosine A2A/biosynthesis , Receptor, Adenosine A2A/genetics , Spodoptera , Time FactorsABSTRACT
BACKGROUND: Anti-CD154 monoclonal antibodies (mAbs) cause long-term graft survival in preclinical allotransplantation experiments. This is the first report on the efficacy and safety of ABI793, a novel human anti-human CD154 mAb, in Cynomolgus renal transplant recipients. METHODS: ABI793 (human immunoglobulin-G1:kappa) was derived from a hybridoma generated after immunization of human immunoglobulin transgenic mice (HuMAb-Mouse, Medarex Inc., Annandale, NJ). Cynomolgus monkey recipients of major histocompatibility complex-mismatched, life-supporting renal allografts were treated repeatedly with intravenous ABI793 for a 3-month period posttransplantation. Graft function was monitored by serum creatinine, and rejection was confirmed histologically. RESULTS: ABI793 binds to human, Cynomolgus and Rhesus monkey CD154; it inhibits dose dependently in vitro CD154:CD40 binding and human mixed lymphocyte reaction. ABI793 is comparable to the mouse anti-human CD154 mAbs 5c8 and 24-31 with respect to affinity, inhibitory capacity, and species specificity; however, ABI793 binds to a different CD154 epitope. With 20 mg/kg of ABI793, five of nine recipients showed substantially prolonged graft survival after cessation of treatment, whereas four of nine recipients were killed because of high serum creatinine while still receiving treatment. ABI793 treatment was associated with episodes of severe acute tubular necrosis (which was unrelated to rejection and responded to fluid and diuretic treatment) and a decrease in platelet numbers. Chronic and acute thromboembolic vascular lesions with hemorrhages were observed in the lung and brain of two allograft recipients. None of these side effects were observed in animals that underwent autotransplantation, thus excluding direct toxicity of ABI793. CONCLUSIONS: ABI793 treatment effectively prevents graft rejection in Cynomolgus monkeys. Evidence for rare thromboembolic events, as also previously observed with different anti-human CD154 mAbs, suggests that thromboembolic complications may be a class effect of anti-CD154 mAbs, unrelated to their epitope specificity.