Reducing recombinant protein expression during CHO pool selection enhances frequency of high-producing cells.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for industrial-scale production of monoclonal antibodies (mAbs) and other protein biologics. Isolation of rare high-producing CHO cell lines from heterogeneous populations of stable transfectants is a daunting task and delays the process of manufacturing of novel biologics. A variety of factors that contribute to the low frequency of high-producing clones have been described; however, the impact of metabolic burden and other stresses (eg.

Enhanced anti-metastatic bioactivity of an IGF-TRAP re-engineered to improve physicochemical properties.

The insulin-like growth factor (IGF) axis has been implicated in the progression of malignant disease and identified as a clinically important therapeutic target. Several IGF-1 receptor (IGF-1R) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signalling. We previously reported on the production of a soluble fusion protein consisting of the extracellular domain of human IGF-1R fused to the Fc portion of human IgG (first generation IGF-TRAP) that bound human IGF-1 and IGF-2 with a 3 log higher affinity than insulin.

Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch.

To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHO) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHO pools producing high levels of recombinant proteins.

The IGF-Trap: Novel Inhibitor of Carcinoma Growth and Metastasis.

The IGFI receptor promotes malignant progression and has been recognized as a target for cancer therapy. Clinical trials with anti-IGFIR antibodies provided evidence of therapeutic efficacy but exposed limitations due in part to effects on, and the compensatory function of, the insulin receptor system. Here, we report on the production, characterization, and biologic activity of a novel, IGF-targeting protein (the IGF-Trap) comprising a soluble form of hIGFIR and the Fc portion of hIgG1.

Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.

E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA).

Flagellin in fusion with human rotavirus structural proteins exerts an adjuvant effect when delivered with replicating but non-disseminating adenovectors through the intrarectal route.

Human rotavirus (HRV) is the worldwide leading cause of gastroenteritis in young children. Two live attenuated HRV vaccines have been approved since 2006. However, these live vaccines still have potential risks including reversion of virulence.

Immunogenic and protective properties of GP5 and M structural proteins of porcine reproductive and respiratory syndrome virus expressed from replicating but nondisseminating adenovectors.

Porcine reproductive and respiratory syndrome virus (PRRSV) is responsible for significant economic losses in the porcine industry. Currently available commercial vaccines do not allow optimal and safe protection. In this study, replicating but nondisseminating adenovectors (rAdV) were used for the first time in pigs for vaccinal purposes.

Expression of human rotavirus chimeric fusion proteins from replicating but non disseminating adenovectors and elicitation of rotavirus-specific immune responses in mice.

The aim of this study was to evaluate the usefulness of replicating but non disseminating adenovirus vectors (AdVs) as vaccine vector using human rotavirus (HRV) as a model pathogen. HRV VP7, VP4, or VP4Δ (N-terminal 336 amino acids of VP4) structural proteins as well as the VP4Δ::VP7 chimeric fusion protein were expressed in mammalian cells when delivered with the AdVs. A preliminary experiment demonstrated that VP4Δ was able to induce a HRV-specific IgG response in BALB/c mice inoculated intramuscularly with AdVs expressing the rotaviral protein.

Efficacy and site-specificity of adenoviral vector integration mediated by the phage φC31 integrase.

Adenoviral vectors deleted of all their viral genes (helper-dependent [HD]) are efficient gene-transfer vehicles. Because transgene expression is rapidly lost in actively dividing cells, we investigated the feasibility of using phage φC31 integrase (φC31-Int) to integrate an HD carrying an attB site and the puromycin resistance gene into human cells (HeLa) and murine myoblasts (C2C12) by co-infection with a second HD-expressing φC31-Int. Because the HD genome is linear, we also investigated whether its circularization, through expression of Cre using a third HD, affects integration.

Strength and muscle specificity of a compact promoter derived from the slow troponin I gene in the context of episomal (gutless adenovirus) and integrating (lentiviral) vectors.

Background: Gutless adenovirus (helper-dependent adenoviral vector; HDAd) and lentiviral vectors (LV) are attractive vectors for the gene therapy of muscle diseases. Because the organization of their DNA (episomal versus integrated) differs, we investigated whether the strength and specificity of ΔUSEx3, a novel muscle-specific promoter previously tested with plasmid, were maintained in the context of these vectors.

Methods: Two HDAds expressing β-galactosidase regulated by ΔUSEx3 or CAG [cytomegalovirus (CMV) enhancer/β-actin promoter], and three LV expressing green fluorescent protein regulated by ΔUSEx3, CMV or a modified skeletal α-actin promoter (SPcΔ5-12), were constructed.

Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system.

Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor β (TGF-β) superfamily, plays important roles in the development of various tissues and organs in mouse and human. In particular, BMP7 is critical for the formation of the nervous system and it is considered to have therapeutic potential in brain injury and stroke. One approach to make BMP7 more suitable for therapeutic purposes is the development of efficient vectors that allow the consistent, reliable and cost-effective production of the BMP7 protein.

Different sites of xenoantigen delivery lead to a virally induced late-onset hepatitis in mice through molecular mimicry.

Background: Epidemiological and laboratory evidences led to the hypothesis that molecular mimicry between viruses and self-proteins could be linked to the onset of autoimmune hepatitis (AIH). Hepatotropic viruses could be good candidates, as a pro-inflammatory environment may facilitate the development of AIH.

Aims: The aims of this study were to test a virus ability to induce an AIH through molecular mimicry and the influence of hepatic inflammation in this process.

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8.

We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha- (TNFα) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellular-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-κB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8.

Novel, versatile, and tightly regulated expression system for Escherichia coli strains.

A novel tightly regulated gene expression system was developed for Escherichia coli by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-isopropylbenzoate (cumate) as an inducer. This novel expression system, referred to as the cumate gene switch, includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the Pseudomonas-based synthetic operator and the cymR repressor protein-encoding gene designed to express constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer (cumate), which is nontoxic to the culture, water soluble, and inexpensive.

High-level recombinant protein production in CHO cells using lentiviral vectors and the cumate gene-switch.

Fast and efficient production of recombinant proteins for structural and functional studies is a crucial issue for research and for industry. To this end, we have developed an efficient system to generate in less than 2 months, starting from the cDNA, pools of CHO cells stably expressing high-level of recombinant proteins. It is based on lentiviral vectors (LVs) for stable transduction coupled with the cumate gene-switch for inducible and efficient gene expression.

Strong muscle-specific regulatory cassettes based on multiple copies of the human slow troponin I gene upstream enhancer.

High-level tissue-specific expression of recombinant proteins in muscle is an important issue for several therapeutic applications. To achieve this goal, we generated several constructs containing one to five copies of the upstream enhancer (USE) of 160-bp of the human slow troponin I gene, linked to that gene's minimal promoter. We also tested constructs made with one to four copies of a 100-bp deletion of USE (DeltaUSE) reported to drive pan-muscle-specific expression in transgenic mice.

Production and characterization of the recombinant Islet Neogenesis Associated Protein (rINGAP).

Islet Neogenesis Associated Protein (INGAP) is implicated in pancreatic islet neogenesis. INGAP peptide, a pentadecapeptide comprising amino acids 104-118, reverses diabetes in rodents and improves glucose homeostasis in patients with diabetes. The mechanism of INGAP action is unknown, but such studies would benefit from the availability of the full-length recombinant protein (rINGAP).

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins.

Background: Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.

Escherichia coli expression and refolding of E/K-coil-tagged EGF generates fully bioactive EGF for diverse applications.

Heterodimerizing peptides, such as the de novo designed E5/K5 peptide pair, have several applications including as tags for protein purification or immobilization. Recently, we demonstrated that E5-tagged epidermal growth factor (EGF), when bound to a K4 expressing adenovirus, promotes retargeting of the adenovirus to EGFR expressing target cells. In this study, we present the Escherichia coli expression, refolding and purification of human EGF fused with the E5-coil (E5-coil-EGF) or with the K5-coil (K5-coil-EGF).

Cellular origins of adult human islet in vitro dedifferentiation.

Cultured human islets can be dedifferentiated to duct-like structures composed mainly of cytokeratin+ and nestin+ cells. Given that these structures possess the potential to redifferentiate into islet-like structures, we sought to elucidate their specific cellular origins. Adenoviral vectors were engineered for beta-, alpha-, delta- or PP-cell-specific GFP expression.

A ligand-pseudoreceptor system based on de novo designed peptides for the generation of adenoviral vectors with altered tropism.

Background: Delivery of transgenes into specific tissues by adenovirus vectors (AdVs) relies on ablations of their natural tropism and on introduction of a new tropism. If the interaction with its natural receptor is ablated, a new packaging cell line is required to produce the AdV. In the present study, we have used two de novo designed peptides (E-Coil and K-Coil) that interact with each other with high affinity to establish a new receptor-ligand system for the propagation of retargeted AdVs.

Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G).

High-level recombinant protein production in CHO cells using an adenoviral vector and the cumate gene-switch.

To facilitate and accelerate the production of eukaryotic proteins with correct post-translational modifications, we have developed a protein production system based on the transduction of Chinese hamster ovary (CHO) cells using adenovirus vectors (AdVs). We have engineered a CHO cell line (CHO-cTA) that stably expresses the transactivator (cTA) of our newly developed cumate gene-switch transcription system. This cell line is adapted to suspension culture and can grow in serum-free and protein-free medium.