Large-Scale Transient Transfection of Suspension Mammalian Cells for VLP Production

ABSTRACTTherapeutic monoclonal antibodies (mAbs) represent one of the most important classes of pharmaceutical proteins to treat human diseases. Most are produced in cultured mammalian cells which is expensive, limiting their availability. Goats, striking a good balance between a relatively short generation time and copious milk yield, present an alternative platform for the cost-effective, flexible, large-scale production of therapeutic mAbs. Here, we focused on cetuximab, a mAb against epidermal growth factor receptor, that is commercially produced under the brand name Erbitux and approved for anti-cancer treatments. We generated several transgenic goat lines that produce cetuximab in their milk. Two lines were selected for detailed characterization. Both showed stable genotypes and cetuximab production levels of up to 10g/L. The mAb could be readily purified and showed improved characteristics compared to Erbitux. The goat-produced cetuximab (gCetuximab) lacked a highly immunogenic epitope that is part of Erbitux. Moreover, it showed enhanced binding to CD16 and increased antibody-dependent cell-dependent cytotoxicity compared to Erbitux. This indicates that these goats produce an improved cetuximab version with the potential for enhanced effectiveness and better safety profile compared to treatments with Erbitux. In addition, our study validates transgenic goats as an excellent platform for large-scale production of therapeutic mAbs.

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Large–Scale Production of Proteins from Mammalian Cells

Nature Biotechnology ◽

10.1038/nbt0588-518 ◽

1988 ◽

Vol 6 (5) ◽

pp. 518-523 ◽

Cited By ~ 20

Author(s):

Malcolm Rhodes ◽

John Birch

Keyword(s):

Mammalian Cells ◽

Large Scale ◽

Scale Production ◽

Large Scale Production

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Insect Cecropins, Antimicrobial Peptides with Potential Therapeutic Applications

International Journal of Molecular Sciences ◽

10.3390/ijms20235862 ◽

2019 ◽

Vol 20 (23) ◽

pp. 5862 ◽

Cited By ~ 16

Author(s):

Daniel Brady ◽

Alessandro Grapputo ◽

Ottavia Romoli ◽

Federica Sandrelli

Keyword(s):

Infectious Diseases ◽

Antimicrobial Peptides ◽

Mammalian Cells ◽

Large Scale ◽

Scale Production ◽

Gram Negative ◽

Social Burden ◽

Large Scale Production ◽

Conventional Antibiotics ◽

The Cost

The alarming escalation of infectious diseases resistant to conventional antibiotics requires urgent global actions, including the development of new therapeutics. Antimicrobial peptides (AMPs) represent potential alternatives in the treatment of multi-drug resistant (MDR) infections. Here, we focus on Cecropins (Cecs), a group of naturally occurring AMPs in insects, and on synthetic Cec-analogs. We describe their action mechanisms and antimicrobial activity against MDR bacteria and other pathogens. We report several data suggesting that Cec and Cec-analog peptides are promising antibacterial therapeutic candidates, including their low toxicity against mammalian cells, and anti-inflammatory activity. We highlight limitations linked to the use of peptides as therapeutics and discuss methods overcoming these constraints, particularly regarding the introduction of nanotechnologies. New formulations based on natural Cecs would allow the development of drugs active against Gram-negative bacteria, and those based on Cec-analogs would give rise to therapeutics effective against both Gram-positive and Gram-negative pathogens. Cecs and Cec-analogs might be also employed to coat biomaterials for medical devices as an approach to prevent biomaterial-associated infections. The cost of large-scale production is discussed in comparison with the economic and social burden resulting from the progressive diffusion of MDR infectious diseases.

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Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014831 ◽

2020 ◽

Vol 295 (50) ◽

pp. 16906-16919

Author(s):

Jae-Hong Kim ◽

Yeojin Seo ◽

Myungjin Jo ◽

Hyejin Jeon ◽

Young-Seop Kim ◽

...

Keyword(s):

Signal Transduction ◽

Cell Migration ◽

Intracellular Signaling ◽

Mammalian Cells ◽

Drug Targets ◽

Large Scale ◽

Genetic Interaction ◽

Genetic Interactions ◽

Signal Transduction Pathways ◽

Global View

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

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A Serum-Free, Transient Transfection System for Enhancing Production of Recombinant Antibodies in Mammalian Cells

Cells and Culture ◽

10.1007/978-90-481-3419-9_39 ◽

2010 ◽

pp. 229-232

Author(s):

Gaurav Backliwal ◽

Sarah Wulhfard ◽

Fanny Delegrange ◽

Lucia Baldi ◽

Maria de Jesus ◽

...

Keyword(s):

Mammalian Cells ◽

Recombinant Antibodies ◽

Transient Transfection ◽

Serum Free ◽

Enhancing Production

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Large Scale Transient Gene Expression in Mammalian Cells

New Developments and New Applications in Animal Cell Technology ◽

10.1007/0-306-46860-3_19 ◽

2006 ◽

pp. 113-116

Author(s):

E. -J. Schlaeger ◽

K. Christensen ◽

G. Schmid ◽

N. Schaub ◽

B. Wipf ◽

...

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Large Scale ◽

Transient Gene Expression ◽

Expression In Mammalian Cells

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Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

Molecular and Cellular Biology ◽

10.1128/mcb.21.22.7807-7816.2001 ◽

2001 ◽

Vol 21 (22) ◽

pp. 7807-7816 ◽

Cited By ~ 154

Author(s):

Shicheng Yang ◽

Stephen Tutton ◽

Eric Pierce ◽

Kyonggeun Yoon

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Es Cells ◽

Embryonic Stem ◽

Transient Transfection ◽

Interferon Response ◽

Green Fluorescent Protein Gene ◽

Double Stranded Rna ◽

Rnai Activity

ABSTRACT Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

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