Mammalian Expression System Research Articles

Abstract 2443: Optides (optimized peptides), a new knottin-based cancer drug discovery platform

Abstract Some of the highest value targets in cancer therapeutics involve protein:protein interactions that are difficult to inhibit with small molecule therapeutics. Peptide drug candidates offer an alternative, but many peptide drugs have poor pharmacokinetic properties and some are immunogenic. Knottin peptides have long been promoted as scaffolds for human drug discovery efforts because the exquisitely tight knot formed by disulfide bridges creates resistance to proteolytic and other forms of degradation. Furthermore, some knottins cross the blood brain barrier, the gut wall, or cell membranes which is ideal for certain targets. Unfortunately, creating knottins synthetically or in bacteria results in a large number of disulfide isomers, which has hampered research. We have created a mammalian expression system that enables production of most knottin scaffolds and variants of native knottins encoded in plant and animal genomes. The platform routinely produces approximately 10 mg/liter of near pure naked peptide, which is sufficient to conduct in vivo studies without major scale-up. The system is endotoxin free. In addition to building the platform, we identified over 200,000 new potential knottin peptides in various genomes, creating a rich collection of scaffolds from which to establish diversity libraries of native and optimized variants. These candidates have potential for targeting therapeutics to cancer cells as a solid tumor penetrating alternative to antibody drug conjugates. Optides also have potential to be developed as anti-cancer drugs themselves. The fact that knottin peptides are not substrates for resistance mechanisms such as p-glycoprotein pumps underscores the potential importance of this class of drugs for those with previously treated cancer. Citation Format: James M. Olson, Roland Strong, Christopher Mehlin, Colin Correnti. Optides (optimized peptides), a new knottin-based cancer drug discovery platform. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2443. doi:10.1158/1538-7445.AM2015-2443

Expression of the human cytomegalovirus pentamer complex for vaccine use in a CHO system.

Human cytomegalovirus (HCMV) causes significant disease worldwide. Multiple HCMV vaccines have been tested in man but only partial protection has been achieved. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of neutralizing antibodies in HCMV seropositive individuals and raises high titers of neutralizing antibodies in small animals and non-human primates (NHP). Thus, Pentamer is a promising candidate for an effective HCMV vaccine. Development of a Pentamer-based subunit vaccine requires expression of high amounts of a functional and stable complex. We describe here the development of a mammalian expression system for large scale Pentamer production. Several approaches comprising three different CHO-originated cell lines and multiple vector as well as selection strategies were tested. Stable cell pools expressed the HCMV Pentamer at a titer of approximately 60 mg/L at laboratory scale. A FACS-based single cell sorting approach allowed selection of a highly expressing clone producing Pentamer at the level of approximately 400 mg/L in a laboratory scale fed-batch culture. Expression in a 50 L bioreactor led to the production of HCMV Pentamer at comparable titers indicating the feasibility of further scale-up for manufacturing at commercial scale. The CHO-produced HCMV Pentamer bound to a panel of human neutralizing antibodies and raised potently neutralizing immune response in mice. Thus, we have generated an expression system for the large scale production of functional HCMV Pentamer at high titers suitable for future subunit vaccine production.

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

Antibody-based therapeutics have now had success in the clinic. The affinity and specificity of the antibody for the target ligand determines the specificity of therapeutic delivery and off-target side effects. The discovery and optimization of high-affinity antibodies to important therapeutic targets could be significantly improved by the availability of a robust, eukaryotic display technology comparable to phage display that would overcome the protein translation limitations of microorganisms. The use of eukaryotic cells would improve the diversity of the displayed antibodies that can be screened and optimized as well as more seamlessly transition into a large-scale mammalian expression system for clinical production. In this study, we demonstrate that the replication and polypeptide display characteristics of a eukaryotic retrovirus, avian leukosis virus (ALV), offers a robust, eukaryotic version of bacteriophage display. The binding affinity of a model single-chain Fv antibody was optimized by using ALV display, improving affinity >2,000-fold, from micromolar to picomolar levels. We believe ALV display provides an extension to antibody display on microorganisms and offers virus and cell display platforms in a eukaryotic expression system. ALV display should enable an improvement in the diversity of properly processed and functional antibody variants that can be screened and affinity-optimized to improve promising antibody candidates.

Functionalization of Bispecific Therapeutic Antibodies Based on Protein Engineering

Although antibodies have been used as molecularly targeted agents for difficult-to-treat diseases such as cancers, the high production costs associated with mammalian expression systems continue to be a drawback. In addition, the clinical efficacy of conventional IgG antibodies is limited. Several types of recombinant antibody (e.g. fused with anticancer drugs, multivalent, or multispecific) have been designed in efforts to develop next-generation antibodies with higher functionality. We used protein engineering to construct several anticancer recombinant antibodies by developing bispecific antibodies that induced specific antitumor effects against cancer cells through the recruitment of lymphocytes. We found that a humanized small bispecific antibody (Ex3) that targets epidermal growth factor receptor on tumor cells and CD3 on T lymphocytes had marked anticancer activity. Furthermore, the function of Ex3 was enhanced by fusion with the human Fc region, domain rearrangement, multimerization, and affinity maturation; a combination of these modifications showed at least additive cytotoxic effects. Interestingly, merely rearranging the domain order of Ex3 induced substantial cytotoxic enhancements, even though the structural format remained the same. Here, we describe our efforts to develop highly functional bispecific antibodies as next-generation therapeutic antibodies using protein engineering.

The production of KIR–Fc fusion proteins and their use in a multiplex HLA class I binding assay

Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR–Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR–Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I.

Relationship between length variations in Ser/Asp-rich repeats in phosphophoryn and in vitro precipitation of calcium phosphate

ObjectivePhosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). PP which contains tandem serine/asparatic acid rich repeats (SDrr) is known to enhance dentin mineralization. The nucleotide sequences coding SDrr are identified in the DSPP genes of toothed animals and the length variations of SDrr between intra- and inter-species have been reported. However, it remains unknown about the relationship between the length variations in SDrr and the functions of PP in matrix mineralization. DesignBy utilizing a mammalian expression system, we generated several recombinant PP proteins (rPP) containing SDrr of different lengths and analyzed their effects on the precipitation of calcium phosphate with an in vitro gel diffusion system. ResultsrPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr, which possessed shortened SDrr that accounted for 62.4 and 36.5% the length of SDrr in full-length rPP (rPP-full), respectively, induced the precipitation of calcium phosphate similar to that of rPP-full at the same molar concentration, whereas rPP-ΔSDrr, in which SDrr were flipped, did not. Furthermore, rPP-Δ63.5 SDrr significantly increased the accumulation of calcium compared with rPP-full at adjusted concentrations so that the same amounts of SDrr were embedded. The results of an ELISA analysis indicated that the amounts of rPP-Δ37.6 SDrr and rPP-Δ63.5 SDrr secreted from transfected cells were 5.2- and 7.1-fold greater than that of rPP-full, respectively. ConclusionsThe generated rPP-Δ63.5 SDrr which can be substituted for rPP-full may be a candidate for a therapeutic molecule to facilitate hard tissue generation such as reparative dentin formation.

Structural Constraints Determine the Glycosylation of HIV-1 Envelope Trimers.

A highly glycosylated, trimeric envelope glycoprotein (Env) mediates HIV-1 cell entry. The high density and heterogeneity of the glycans shield Env from recognition by the immune system, but paradoxically, many potent broadly neutralizing antibodies (bNAbs) recognize epitopes involving this glycan shield. To better understand Env glycosylation and its role in bNAb recognition, we characterized a soluble, cleaved recombinant trimer (BG505 SOSIP.664) that is a close structural and antigenic mimic of native Env. Large, unprocessed oligomannose-type structures (Man8-9GlcNAc2) are notably prevalent on the gp120 components of the trimer, irrespective of the mammalian cell expression system or the bNAb used for affinity purification. In contrast, gp41 subunits carry more highly processed glycans. The glycans on uncleaved, non-native oligomeric gp140 proteins are also highly processed. A homogeneous, oligomannose-dominated glycan profile is therefore a hallmark of a native Env conformation and a potential Achilles' heel that can be exploited for bNAb recognition and vaccine design.

Role of cytochrome P450 2C8*3 (CYP2C8*3) in paclitaxel metabolism and paclitaxel-induced neurotoxicity.

The CYP2C8*3 allele has been suggested as a risk factor for paclitaxel-induced neuropathy but the data hitherto published are conflicting. In total 435 patients were investigated with respect to maximum neuropathy grade and accumulated paclitaxel dose. The enzymatic properties of CYP2C8.3 variant were analyzed using heterologous mammalian HEK293 cell expression system. No significant association between CYP2C8*3 allele and neuropathy was found, although a trend was observed. The paclitaxel and amodiaquine metabolism by CYP2C8.3 were found similar to CYP2C8.1, whereas CYP2C8.3 was more efficient in the metabolism of rosiglitazone. These results indicate a difference in substrate specificity between CYP2C8.1 and CYP2C8.3; however, the CYP2C8*3 allele has no major impact on paclitaxel metabolism in vitro or of paclitaxel-induced neuropathy in vivo. Original submitted on 6 February 2015; revision submitted on 9 April 2015.

Versatility of Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assays for Biologics Drug Discovery

Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

0441 : Electrophysiological characterization of a novel SCN5A mutation causing Brugada syndrome, using cardiomyocytes differentiated from hiPSCs

Brugada syndrome is a human hereditary cardiac disease known to cause ventricular tachyarrhythmias (torsade de pointes) that can lead to sudden death. In about 20% of the Brugada affected patients, mutations have been identified in the gene encoding the Na+ ion channel, SCN5A. Up to now, genotype-phenotype studies have been performed using heterologous expression systems that lack the correct cellular context of a cardiomyocyte. Human Induced Pluripotent stem cells (hiPSCs) offer a now new paradigm for gene mutation characterization. In this study, using cardiomyocytes differentiated from hiPSCs, we have electrophysiologically characterized a previously undescribed mutation in SCN5A gene, carried by a Brugada affected patient. hiPSCs from a Brugada affected patient carrying the N1722D mutation in SCN5A have been generated and validated. hiPSCs from a healthy subject were used as control. Using patch clamp techniques, the biophysical properties of the Na+ channel and action potential characteristics were evaluated in both cardiomyocytes differentiated from these hiPSCs and in a mammalian expression system expressing the mutant channel. Preliminary data from both cellular models suggest a three-times reduction in Na+ current. The hiPSCs-derived cardiomyocytes revealed a specific action potential phenotype which is still under investigation Brugada syndrome modeling using hiPSCs-derived cardiomyocytes suggests that this cellular model recapitulates the characteristics of a loss-of-function Na+ channel mutation and that hiPSCs-derived cardiomyocytes can be used as an accurate model for cardiac Na+ channel disease.

Structural Analysis of Dopamine- and Amphetamine-Induced Depolarization Currents in the Human Dopamine Transporter

Amphetamine (AMPH) induces depolarizing currents through the human dopamine transporter (hDAT). Recently we discovered that the S(+) enantiomer of AMPH induces a current through hDAT that persists long after its removal from the external milieu. The persistent current is less prominent for R(-)AMPH and essentially absent for dopamine (DA)-induced currents. Related agents such as methamphetamine also exhibit persistent currents, which are present in both frog oocyte and mammalian HEK expression systems. Here, we study hDAT-expressing Xenopus laevis oocytes voltage-clamped and exposed from outside to DA, S(+)AMPH, R(-)AMPH, and related synthesized compounds, including stereoisomers. The goal of the study was to determine how structural transitioning from dopamine to amphetamine influences hDAT potency and action. At saturating concentrations, S(+)AMPH or R(-)AMPH induce a sharply rising depolarizing current from -60 mV that is comparable in amplitude to DA-induced currents. The magnitude and duration of the currents and the presence or absence of persistent currents depend on the concentration, duration of exposure, and chemical structure and enantiomeric versions of the agents.

Cocktail of H5N1 COBRA HA vaccines elicit protective antibodies against H5N1 viruses from multiple clades

Pandemic outbreaks of influenza are caused by the emergence of a pathogenic and transmissible virus to which the human population is immunologically naïve. Recent outbreaks of highly pathogenic avian influenza (HPAI) of the H5N1 subtype are of particular concern because of the high mortality rate (60% case fatality rate) and novel subtype. In this study, we have engineered an influenza virus-like particle (VLP) that contains a synthetic, consensus-based HA molecule using a new methodology, computationally optimized broadly reactive antigen (COBRA). Three COBRA H5N1 HA proteins have been engineered based upon (1) human clade 2 H5N1 sequences, (2) human and avian clade 2 sequences, and (3) all H5N1 influenza sequences recorded between 2005-2008. Each hemagglutinin protein retained the ability to bind the appropriate receptors, as well as the ability to mediate particle fusion, following purification from a mammalian expression system. COBRA VLP vaccines were administered to mice and the humoral immune responses were compared to those induced by VLPs containing an HA derived from a primary viral isolate. Using a single vaccination (0.6 ug HA dose with an adjuvant) all animals vaccinated with COBRA clade 2 HA H5N1 VLPs had protective levels of HAI antibodies to a representative isolate from each subclade of clade 2, but lower titers against other clades. The addition of avian sequences from other clades expanded breadth of HAI antibodies to the divergent clades, but still not all of the 25 H5N1 viruses in the panel were recognized by antibodies elicited any one H5N1 COBRA VLP vaccine. Vaccination of mice with a cocktail of all 3 COBRA HA VLP vaccines, in a prime-boost regimen, elicited an average HAI titer greater than 1:40 against all 25 viruses. Collectively, our findings indicate that the elicited antibody response following VLP vaccination with all 3 COBRA HA vaccine simultaneously elicited a broadly-reactive set of antibodies that recognized H5N1 viruses from 11 H5N1 clades/subclades isolated over a 12-year span.

A stable human-cell system overexpressing cystic fibrosis transmembrane conductance regulator recombinant protein at the cell surface.

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, and an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTR(FLAG)-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTR(FLAG)-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment future CF drug discovery efforts, including biophysical and structural studies.

Effective isotope labeling of proteins in a mammalian expression system.

Isotope labeling of biologically interesting proteins is a prerequisite for structural and dynamics studies by NMR spectroscopy. Many of these proteins require mammalian cofactors, chaperons, or posttranslational modifications such as myristoylation, glypiation, disulfide bond formation, or N- or O-linked glycosylation; and mammalian cells have the necessary machinery to produce them in their functional forms. Here, we describe recent advances in mammalian expression, including an efficient adenoviral vector-based system, for the production of isotopically labeled proteins. This system enables expression of mammalian proteins and their complexes, including proteins that require posttranslational modifications. We describe a roadmap to produce isotopically labeled (15)N and (13)C posttranslationally modified proteins, such as the outer domain of HIV-1 gp120, which has four disulfide bonds and 15 potential sites of N-linked glycosylation. These methods should allow NMR spectroscopic analysis of the structure and function of posttranslationally modified and secreted, cytoplasmic, or membrane-bound proteins.

Production of Highly Sialylated Recombinant Glycoproteins Using Ricinus communis Agglutinin-I-Resistant CHO Glycosylation Mutants.

The degree of sialylation of therapeutic glycoproteins affects its circulatory half-life and efficacy because incompletely sialylated glycoproteins are cleared from circulation by asialoglycoprotein receptors present in the liver cells. Mammalian expression systems, often employed in the production of these glycoprotein drugs, produce heterogeneously sialylated products. Here, we describe how to produce highly sialylated glycoproteins using a Chinese hamster ovary (CHO) cell glycosylation mutant called CHO-gmt4 with human erythropoietin (EPO) as a model glycoprotein. The protocol describes how to isolate and characterize the CHO glycosylation mutants and how to assess the sialylation of the recombinant protein using isoelectric focusing (IEF). It further describes how to inactivate the dihydrofolate reductase (DHFR) gene in these cells using zinc finger nuclease (ZFN) technology to enable gene amplification and the generation of stable cell lines producing highly sialylated EPO.

Engineering artificial cells by combining HeLa-based cell-free expression and ultrathin double emulsion template.

Generation of artificial cells provides the bridge needed to cover the gap between studying the complexity of biological processes in whole cells and studying these same processes in an in vitro reconstituted system. Artificial cells are defined as the encapsulation of biologically active material in a biological or synthetic membrane. Here, we describe a robust and general method to produce artificial cells for the purpose of mimicking one or more behaviors of a cell. A microfluidic double emulsion system is used to encapsulate a mammalian cell-free expression system that is able to express membrane proteins into the bilayer or soluble proteins inside the vesicles. The development of a robust platform that allows the assembly of artificial cells is valuable in understanding subcellular functions and emergent behaviors in a more cell-like environment as well as for creating novel signaling pathways to achieve specific cellular behaviors.

Strategies for recombinant production of human glycosylation enzymes: Comparison of mammalian and insect cell expression systems

Strategies for recombinant production of human glycosylation enzymes: Comparison of mammalian and insect cell expression systems

Complete clarification solution for processing high density cell culture harvests

Traditional clarification of mammalian cell culture typically combines multiple separation technologies to remove cells in a primary step, and colloids in a secondary step. Recent advances in mammalian expression systems have not only led to cell cultures with very high density and increased protein titers, but also to feed streams with a high proportion of solids and colloids that reduce the efficiency of the separation step. Issues with scale-up, flexibility and installation limit the attractiveness of centrifugation, whereas clarification solely by depth filtration can be costly due to high filter areas required. Therefore, an efficient manufacture process must be developed to overcome these restrictions. In this study we present a complete separation solution that combines cell harvest pretreatment with a polycationic flocculating agent (polydiallyldimethylammonium chloride or pDADMAC), followed by depth filtration using depth filters that were specifically developed for filtration of flocculated or precipitated feed streams. Multiple antibody feed streams treated with pDADMAC and subsequently filtered using pretreatment adapted depth filters resulted in improved removal of cells and colloids, increased clarification throughput, efficient reduction of DNA and high process yield. The residual level of pDADMAC in the filtrates was assessed by surface plasmon resonance spectroscopy. Overall, this next generation clarification solution presented herein led to a robust, high yield, economical separation process with enhanced impurity removal that can be readily incorporated into current clarification platforms.

Uncovering methods for the prevention of protein aggregation and improvement of product quality in a transient expression system.

Mammalian expression systems are used routinely for the production of recombinant proteins as therapeutic molecules as well as research tools. Transient expression has become increasingly popular in recent years due to its rapid timeline and improvements in expression level. While improvements to transient expression systems have focused mainly on the level of protein expression, the aspect of protein quality has received little attention. The removal of undesirable products, such as aggregation, depends primarily on purification, requiring additional cumbersome steps, which can lead to a lower product yield and longer timelines. In this study, we show that reducing the level of transcription by transfecting at a lower gene dose improves the quality of secreted molecules prone to aggregation. For gene dosing to have this effect, it is critical for the carrier DNA to be an empty vector containing the same elements as the gene containing plasmid. This approach can be used in combination with a temperature shift to hypothermic conditions during production to enhance the effect. The observed improvements not only minimized aggregation levels, but also generated products with overall superior quality, including more homogeneous signal peptide cleavage and N-linked glycosylation profiles. These techniques have produced a similar improvement in product quality with a variety of other molecules, suggesting that this may be a general approach to enhance product quality from transient expression systems.

Production of cell surface and secreted glycoproteins in mammalian cells.

Mammalian protein expression systems are becoming increasingly popular for the production of eukaryotic secreted and cell surface proteins. Here we describe methods to produce recombinant proteins in adherent or suspension human embryonic kidney cell cultures, using transient transfection or stable cell lines. The protocols are easy to scale up and cost-efficient, making them suitable for protein crystallization projects and other applications that require high protein yields.