Transient recombinant protein expression in mammalian cells:the role of mRNA level and stability

Transient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt was made to enhance TGE production by increasing the amount of transfected plasmid DNA. However, this approach did not result in increased recombinant protein levels; on the contrary, transfection with an excess of plasmid DNA (> 1.25 μg/ml) had a negative impact on transgene mRNA levels and protein production. Moreover, it was also observed that recombinant protein yield was strongly dependent on the mRNA level. Therefore, three strategies aimed at increasing the amount of transgene mRNA were investigated. For the first approach, transfected cells were exposed to hypothermic conditions during the production phase. It was already known that lower temperatures increase protein production several fold in recombinant CHO DG44-derived cell lines. The second strategy involved the treatment of transfected cells with valproic acid, a histone deacetylase inhibitor that reduces the effects of gene silencing. The third approach aimed to increase transgene mRNA levels by overexpressing transcription factors and growth factors. With the first two strategies recombinant antibody yields of 60-80 mg/L were achieved whereas the untreated control transfections produced only 5-10 mg/L. Combination of the two strategies led to the production of 90 mg/L of antibody. Moreover, in the treated cultures, the steady-state level of transgene mRNA was 3-5 times higher than in the untreated cultures and remained stable up to 6 days post-transfection. Using the third approach, the increase in recombinant protein production was moderate and transgene mRNA amounts were only 2-fold higher in treated samples compared to the control. When specific proteins such as c-fos, c-jun, NF-kB, and acidic fibroblast growth factor (aFGF) were overexpressed the recombinant antibody production was 20 mg/L compared to 5 mg/L for the control transfection. Overexpression of either a transcription factor or a growth factor in combination with treatment with valproic acid allowed the recombinant protein yield to reach 90 mg/L. However, the benefit of the overexpressed factors was minimal compared to the effect of valproic acid alone. In conclusion, it was demonstrated that the level and stability of transgene mRNA are important factors for increasing volumetric yields in transiently transfected CHO DG44 cells. Furthermore, three approaches aimed to increase mRNA amounts were tested. Exposure to hypothermic conditions and treatment with valproic acid were the two best strategies tested. Both are simple, cost-effective, and scalable making transient gene expression in CHO DG44 cells a feasible alternative for rapid production of gram amounts of recombinant protein

A critical evaluation of the tumor-targeting properties of bispecific antibodies based on quantitative biodistribution data

Bispecific and bifunctional antibodies are attracting considerable interest as innovative anti-cancer therapeutics, but their ability to selectively localize at the tumor site has rarely been studied by quantitative biodistribution studies in immunocompetent animal models or in patients. Here, we describe the production of a novel bifunctional antibody, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin) fused to the extracellular portion of CD86 (co-stimulatory molecule B7.2). However, the fusion molecule was unable to target tumors in vivo. These data suggest that bispecific antibodies do not always localize on tumors and should therefore be characterized by imaging or biodistribution studie

Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

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Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems

Cell expansion, gene transfer and protein production were all executed with a single serum-free, animal protein-free commercial medium designed for suspension-adapted Chinese hamster ovary cells (CHO DG44). This is a most important process to consider for clinical production of recombinant proteins. The transfection with polyethylenimine (PEI) was shown here to be scalable using both stirred-tank bioreactors of 3- and 150-l and novel agitated cultivation vessels (50ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2ml to 80l. The maximum transient recombinant antibody yield was 22mg/l, the highest ever reported for a multiliter transfection in CHO. The transiently expressed protein had the same extent of glycosylation as the same antibody produced from a stably transfected recombinant CHO cell lin

Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology

A critical evaluation of the tumor-targeting properties of bispecific antibodies based on quantitative biodistribution data

ISSN:1741-0126ISSN:1741-0134ISSN:0269-2139ISSN:1460-213

Valproic acid enhances recombinant mRNA and protein levels in transiently transfected Chinese hamster ovary cells

Valproic acid (VPA) is a small molecule that inhibits histone deacetylase activity. Here we report that VPA increases recombinant mRNA and protein levels in transiently transfected CHO DG44 cells. In the presence of VPA, transient recombinant antibody yields of up to 40 mg/L were achieved in simple batch cultures. The steady-state levels of the IgG light and heavy chain mRNAs were nearly 10 times higher than in the untreated control transfection even though the level of transfected plasmid DNA was the same in the presence or absence of VPA. The combination of VPA treatment and incubation of the transfected cells in mildly hypothermic conditions resulted in recombinant antibody yields of over 90 mg/L by 6 days post-transfection in batch cultures. The results demonstrated that the treatment of transfected CHO DG44 cells with VPA is a cost-effective strategy for enhancing transient gene expression by increasing the transgene mRNA levels

Potency-matched dual cytokine-antibody fusion proteins for cancer therapy

A novel biopharmaceutical, consisting of the F8 mAb (specific to a splice isoform of fibronectin) simultaneously fused to both TNF and IL2, was found to react with the majority of solid tumors and hematologic malignancies in mouse and man, but not with healthy adult tissues. The product selectively localized to neoplastic lesions in vivo, as evidenced by quantitative biodistribution studies using radioiodinated protein preparations. When the potency of the cytokine payloads was matched by a single-point mutation, the resulting fusion protein (IL2-F8-TNFmut) eradicated soft-tissue sarcomas in immunocompetent mice, which did not respond to individual antibody-cytokine fusion proteins or by standard doxorubicin treatment. Durable complete responses were also observed in mice bearing CT26, C1498, and F9 tumors. The simultaneous delivery of multiple proinflammatory payloads to the cancer site conferred protective immunity against subsequent tumor challenges. A fully human homolog of IL2-F8-TNFmut, which retained selectivity similar to its murine counterpart when tested on human material, may open new clinical applications for the immunotherapy of cancer. Mol Cancer Ther; 16(11); 2442-51. ©2017 AACR

Enhanced therapeutic activity of non-internalizing small-molecule-drug conjugates targeting carbonic anhydrase IX in combination with targeted interleukin-2

Purpose: Antibody-drug conjugates and small-molecule-drug conjugates have been proposed as alternatives to conventional anticancer cytotoxic agents, with the potential to deliver bioactive payloads to the site of disease, helping spare normal tissues.Experimental Design: Here, we describe a novel small-molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product featured a peptidic linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload.Results: A potent anticancer activity was observed in nude mice bearing SKRC-52 renal cell carcinoma xenografts, but no durable complete responses could be observed in this model. However, when the product was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering IL2 to the tumor neovasculature), all treated mice in the combination group could be rendered tumor free, in a process that favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small-molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX.Conclusions: These findings may be of clinical significance, because carbonic anhydrase IX is overexpressed in the majority of clear cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of IL2 helps potentiate the action of targeted cytotoxics, leading to cancer eradication in models that cannot be cured by conventional chemotherapy