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.
Purpose: Interleukin-12 (IL12) is a potent proinflammatory cytokine with antitumor activity. Its heterodimeric nature makes it compatible with a large variety of different immunocytokine formats. Here we report the design, production, and characterization of a novel immunocytokine, based on the fusion of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of tumor neovasculature) with IL12 (termed IL12-F8-F8).Experimental Design: We developed a novel immunocytokine based on the sequential fusion of interleukin-12 as a single polypeptide with two F8 antibodies in single-chain Fv (scFv) format. The fusion protein was characterized in vitro, and its targeting performance was assessed in vivo. The immunocytokine antitumor activity was studied as monotherapy as well as in combination therapies in three different murine tumor models. Moreover, depletion experiments and tumor analysis revealed a dominant role of natural killer cells for the mechanism of action.Results: IL12-F8-F8 can be produced in mammalian cells, yielding a product of good pharmaceutical quality, capable of selective localization on the tumor neovasculature in vivo, as judged by quantitative biodistribution analysis with radioiodinated protein preparations. The protein potently inhibited tumor growth in three different immunocompetent syngeneic models of cancer. The treatment was generally well tolerated. Moreover, the IL12-F8-F8 fusion protein could be produced both with murine IL12 (mIL12) and with human IL12 (hIL12).Conclusions: The potent antitumor activity of mIL12-F8-F8, studied alone or in combination with paclitaxel in different tumor models, paves the way to the clinical development of the fully human immunocytokine. Clin Cancer Res; 18(15); 4092-103. Ó2012 AACR.
Large-scale transient gene expression (TGE) in mammalian cells is a rapid method to generate recombinant proteins, but the volumetric productivity for secreted proteins is still more than an order of magnitude lower than the yields typically achieved with recombinant cell lines. Here transient recombinant protein production in Chinese hamster ovary cells transfected with linear 25 kDa polyethylenimine was significantly enhanced by incubation of the cells at temperatures ranging from 29 to 33°C after DNA delivery. With this approach, transient recombinant antibody yields of 60-80 mg/L were achieved within 6 days of transfection. The increase in TGE correlated with the accumulation of cells in the G1 phase of the cell cycle, increased cell size, higher cell viability, higher steady-state levels of transgene mRNA, reduced consumption of nutrients, and decreased accumulation of waste products. The enhancement of TGE was not vector-dependent, but the presence of the woodchuck hepatitis virus post-transcriptional regulatory element in the 3′ untranslated region of the transgene mRNA increased transient recombinant antibody expression more than 3-fold at 31°C as compared to expression at 37°C. The yields achieved by the low-temperature enhancement of TGE in CHO cells makes this technology feasible for the rapid production of gram amounts of secreted recombinant proteins at large scale (up to 100 L).
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. 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. 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. 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. .
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 , 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-TNF) 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-TNF, which retained selectivity similar to its murine counterpart when tested on human material, may open new clinical applications for the immunotherapy of cancer. .
The development of antibody-drug conjugates (ADC), a promising class of anticancer agents, has traditionally relied on the use of antibodies capable of selective internalization in tumor cells. We have recently shown that also noninternalizing antibodies, coupled to cytotoxic drugs by means of disulfide linkers that can be cleaved in the tumor extracellular environment, can display a potent therapeutic activity. Here, we have compared the tumor-targeting properties, drug release rates, and therapeutic performance of two ADCs, based on the maytansinoid DM1 thiol drug and on the F8 antibody, directed against the alternatively spliced Extra Domain A (EDA) domain of fibronectin.The antibody was used in IgG or in small immune protein (SIP) format. In both cases, DM1 was coupled to unpaired cysteine residues, resulting in a drug-antibody ratio of 2. In biodistribution studies, SIP(F8)-SS-DM1 accumulated in the tumor and cleared from circulation more rapidly than IgG(F8)-SS-DM1. However, the ADC based on the IgG format exhibited a higher tumor uptake at later time points (e.g., 33%IA/g against 8%IA/g at 24 hours after intravenous administration). In mouse plasma, surprisingly, the ADC products in IgG format were substantially more stable compared with the SIP format (half-lives >48 hours and <3 hours at 37 C, respectively), revealing a novel mechanism for the control of disulfide-based drug release rates. Therapy experiments in immunocompetent mice bearing murine F9 tumors revealed that SIP(F8)-SS-DM1 was more efficacious than IgG(F8)-SS-DM1 when the two products were compared either in an equimolar basis or at equal milligram doses.
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 (50 ml ventilated centrifuge tubes and 1-l square-shaped glass bottles) that lack any instrumentation. The transient transfections spanned a range of working volumes from 2 ml to 80 l. The maximum transient recombinant antibody yield was 22 mg/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 line.
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