Optimization of a transient antibody expression platform towards high titer and efficiency.

Transient gene expression (TGE) using mammalian cells is an extensively used technology for the production of antibodies and recombinant proteins and has been widely adopted by both academic and industrial labs. Chinese Hamster Ovary (CHO) cells have become one of the major workhorses for TGE of recombinant antibodies due to their attractive features: post-translational modifications, adaptation to high cell densities, and use of serum-free media. In this study, we describe the optimization of parameters for TGE for antibodies from CHO cells. Through a matrix evaluation of multiple factors including inoculum, transfection conditions, amount and type of DNA used, and post-transfection culture conditions, we arrived at an uniquely optimized process with higher titer and reduced costs and time, thus increasing the overall efficiency of early antibody material supply. We further investigated the amount of coding DNA used in TGE and the influence of kinetics and size of the transfection complex on the in vitro efficiency of the transfection. We present here the first report of an optimized TGE platform using Filler DNA in an early drug discovery setting for the screening and production of therapeutic mAbs.

Changes in Chromosome Counts and Patterns in CHO Cell Lines upon Generation of Recombinant Cell Lines and Subcloning.

Chinese hamster ovary (CHO) cells are the number one production system for therapeutic proteins. A pre-requirement for their use in industrial production of biopharmaceuticals is to be clonal, thus originating from a single cell in order to be phenotypically and genomically identical. In the present study it was evaluated whether standard procedures, such as the generation of a recombinant cell line in combination with selection for a specific and stable phenotype (expression of the recombinant product) or subcloning have any impact on karyotype stability or homogeneity in CHO cells. Analyses used were the distribution of chromosome counts per cell as well as chromosome painting to identify specific karyotype patterns within a population. Results indicate that subclones both of the host and the recombinant cell line are of comparable heterogeneity and (in)stability as the original pool. In contrast, the rigorous selection for a stably expressing phenotype generated cell lines with fewer variation and more stable karyotypes, both at the level of the sorted pool and derivative subclones. We conclude that the process of subcloning itself does not contribute to an improved karyotypic homogeneity of a population, while the selection for a specific cell property inherently can provide evolutionary pressure that may lead to improved chromosomal stability as well as to a more homogenous population.

Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting.

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO-S, CHO-K1 8 mM glutamine, and CHO-K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO-S and CHO-K1, with the diversity increasing and new variants appearing, while CHO-K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.

A single-step FACS sorting strategy in conjunction with fluorescent vital dye imaging efficiently assures clonality of biopharmaceutical production cell lines.

The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation. Within the flow cytometry step, two unit operations are evaluated separately, doublet discrimination during event selection for deposition and droplet deposition accuracy. The imaging procedure is evaluated for the accuracy of detection of non-clonal populations. By employing mixing experiments of cell populations, the authors demonstrate that doublet discrimination is highly efficient, and that an appropriately set up flow cytometry system already can generate >99.5% true single cell clones. The efficiency of the described imaging process depends on several factors, reaching an optimal detection rate of non-clonal wells of about 99.8%. Our results demonstrate that one well characterized cloning step generate biopharmaceutical production cell lines with a probability of clonality of >99.99%.

ATF6ß-based fine-tuning of the unfolded protein response enhances therapeutic antibody productivity of Chinese hamster ovary cells.

The dynamics of protein folding and secretion are key issues in improving the productivity and robustness of Chinese hamster ovary (CHO) producer cells. High recombinant protein secretion in CHO producer clones triggers the activation of the unfolded protein response (UPR), an intracellular response to the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). We previously reported that the human microRNA (miRNA) miR-1287 enhances productivity in IgG-expressing CHO cells (CHO-IgG). Here, through next-generation sequencing (NGS), we identified the activating transcription factor 6 beta (ATF6ß), a repressor of the pro-survival and UPR promoting factor ATF6α, as a direct target gene of miR-1287 in CHO-IgG cells. We show that the transient depletion of ATF6ß resulted in enhanced specific productivity comparable to that of miR-1287-expressing CHO-IgG cells. Strikingly, stable ATF6ß knockdown in CHO-IgG cells significantly improved antibody titer and viable cell density under fed-batch conditions. This was associated with the elevated expression of the UPR genes glucose-regulated protein 78 (GRP78), homocysteine inducible ER protein with ubiquitin like domain 1 (Herpud1) and CCAAT/enhancer-binding protein homologous protein (CHOP). We hence demonstrate that ATF6ß-based cell line engineering is a promising strategy to improve the productivity of CHO producer cells by activating an optimally balanced UPR program. Biotechnol. Bioeng. 2017;114: 1310-1318. © 2017 Wiley Periodicals, Inc.

Secretory pathway optimization of CHO producer cells by co-engineering of the mitosRNA-1978 target genes CerS2 and Tbc1D20.

Chinese Hamster Ovary (CHO) cells are the most commonly used host for the production of biopharmaceuticals. Although transcription and translation engineering strategies have been employed to generate high-producer cell clones, the secretory pathway still remains a bottleneck in cellular productivity. In this study we show that ectopic expression of a human mitochondrial genome-encoded small RNA (mitosRNA-1978) in an IgG expressing CHO cell line strongly improved specific productivity by functioning in a microRNA-like fashion. By next generation sequencing we identified two endoplasmic reticulum (ER)-localized proteins, Ceramide Synthase 2 (CerS2) and the Rab1 GAP Tbc domain family member 20 (Tbc1D20), as target genes of mitosRNA-1978. Combined transient siRNA-mediated knockdown of CerS2 and Tbc1D20 resulted in increased specific productivity of CHO-IgG cells, thus recapitulating the mitosRNA-1978 phenotype. In support of a function in vesicular trafficking at the level of the ER, we provide evidence for altered cellular ceramide composition upon CerS2 knockdown and increased activity of Rab1 in CHO-IgG cells depleted of Tbc1D20. Importantly, in a fed-batch process, the combined stable knockdown of CerS2 and Tbc1D20 in CHO-IgG cells resulted in dramatically increased antibody production which was accompanied by enhanced cell growth. Thus, by identifying mitosRNA-1978 target genes in combination with an informed shRNA-mediated co-engineering approach we successfully optimized the secretory capacity of CHO producer cells used for the manufacturing of therapeutic proteins.

RUN and FYVE domain-containing protein 4 enhances autophagy and lysosome tethering in response to Interleukin-4.

Autophagy is a key degradative pathway coordinated by external cues, including starvation, oxidative stress, or pathogen detection. Rare are the molecules known to contribute mechanistically to the regulation of autophagy and expressed specifically in particular environmental contexts or in distinct cell types. Here, we unravel the role of RUN and FYVE domain-containing protein 4 (RUFY4) as a positive molecular regulator of macroautophagy in primary dendritic cells (DCs). We show that exposure to interleukin-4 (IL-4) during DC differentiation enhances autophagy flux through mTORC1 regulation and RUFY4 induction, which in turn actively promote LC3 degradation, Syntaxin 17-positive autophagosome formation, and lysosome tethering. Enhanced autophagy boosts endogenous antigen presentation by MHC II and allows host control of Brucella abortus replication in IL-4-treated DCs and in RUFY4-expressing cells. RUFY4 is therefore the first molecule characterized to date that promotes autophagy and influences endosome dynamics in a subset of immune cells.

Induction of GADD34 is necessary for dsRNA-dependent interferon-ß production and participates in the control of Chikungunya virus infection.

Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection.

Autophagy inhibition promotes defective neosynthesized proteins storage in ALIS, and induces redirection toward proteasome processing and MHCI-restricted presentation.

A significant portion of newly synthesized protein fails to fold properly and is quickly degraded. These defective ribosomal products (DRiPs) are substrates for the ubiquitin-proteasome system (UPS) and give rise to a large fraction of peptides presented by major histocompatibility complex class I molecules (MHCI). Here, we showed that DRiPs are also autophagy substrates, which accumulate upon autophagy inhibition in aggresome-like-induced structures (ALIS). Aggregation is critically depending on p62/SQSTM1, but occurs in the absence of activation of the NRF2 signaling axis and transcriptional regulation of p62/SQSTM1. We demonstrated that autophagy-targeted DRiPs can become UPS substrates and give rise to MHCI presented peptides upon autophagy inhibition. We further demonstrated that autophagy targeting of DRiPs is controlled by NBR1, but not p62/SQSTM1, CHIP or BAG-1. Active autophagy therefore directly modulates MHCI presentation by constantly degrading endogenous defective neosynthesized antigens, which are submitted to at least two distinct quality control mechanisms.

Protein phosphatase 1 subunit Ppp1r15a/GADD34 regulates cytokine production in polyinosinic:polycytidylic acid-stimulated dendritic cells.

In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation that exhibits specific mechanisms to control the immune response. Here we show that in response to polyriboinosinic:polyribocytidylic acid (pI:C), DCs mount a specific integrated stress response during which the transcription factor ATF4 and the growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), a phosphatase 1 (PP1) cofactor, are expressed. In agreement with increased GADD34 levels, an extensive dephosphorylation of the translation initiation factor eIF2α was observed during DC activation. Unexpectedly, although DCs display an unusual resistance to protein synthesis inhibition induced in response to cytosolic dsRNA, GADD34 expression did not have a major impact on protein synthesis. GADD34, however, was shown to be required for normal cytokine production both in vitro and in vivo. These observations have important implications in linking further pathogen detection with the integrated stress response pathways.

BAD-LAMP is a novel biomarker of nonactivated human plasmacytoid dendritic cells.

The brain and dendritic cell (BAD)-associated lysosome-associated membrane protein (LAMP)-like molecule (BAD-LAMP, c20orf103, UNC-46) is a newly identified member of the family of LAMPs. BAD-LAMP expression in the mouse is confined to neurons. We demonstrate here that in humans, BAD-LAMP can specifically be found in the type I IFN-producing plasmacytoid dendritic cells (pDCs). Human BAD-LAMP is localized in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of freshly isolated CD123(+) pDCs and is rapidly lost upon activation by unmethylated cytosine-phosphate-guanine (CpG) oligonucleotides. The restricted pattern of BAD-LAMP expression allows for the rapid identification of normal and leukemic human pDCs in tissues and blood.

Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction.

TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.

The alkaloid emetine as a promising agent for the induction and enhancement of drug-induced apoptosis in leukemia cells.

Emetine, a natural alkaloid from Psychotria ipecacuanha, has been used in phytomedicine to induce vomiting, and to treat cough and severe amoebiasis. Certain data suggest the induction of apoptosis by emetine in leukemia cells. Therefore, we examined the suitability of emetine for the sensitisation of leukemia cells to apoptosis induced by cisplatin. In response to emetine, we found a strong reduction in viability, an induction of apoptosis and caspase activity comparable to the cytotoxic effect of cisplatin. Moreover, emetine had an additive effect and increased cisplatin-induced apoptosis. Mechanistically, we demonstrate by DNA array analysis that emetine alone or together with cisplatin down-regulates several anti-survival genes and up-regulates several pro-apoptotic signalling molecules along with other effects on signalling. These data show that emetine is a strong inducer of apoptosis in leukemia cells and could be a suitable cytotoxic drug alone or in combination with other chemotherapeutics to sensitise leukemia cells to apoptosis.

Clinical and mechanistic aspects of glucocorticoid-induced chemotherapy resistance in the majority of solid tumors.

BACKGROUND: Glucocorticoids have been used widely in conjunction with cancer therapy due to their ability to induce apoptosis in hematological cells and to prevent nausea and emesis. However, recent data including ours, suggest induction of therapy-resistance by glucocorticoids in solid tumors, although it is unclear whether this happens only in few carcinomas or is a more common cell type specific phenomenon. MATERIAL AND METHODS: We performed an overall statistical analysis of our new and recent data with 157 tumor probes evaluated in vitro, ex vivo and in vivo. The effect of glucocorticoids on apoptosis, viability and cell cycle progression under diverse clinically important questions was examined. RESULTS: New in vivo results demonstrate glucocorticoid-induced chemotherapy resistance in xenografted prostate cancer. In an overall statistical analysis we found glucocorticoid-induced resistance in 89% of 157 analysed tumor samples. Resistance is common for several cytotoxic treatments and for several glucocorticoid-derivatives and due to an inhibition of apoptosis, promotion of viability and cell cycle progression. Resistance occurred at clinically achievable peak plasma levels of patients under anti-emetic glucocorticoid therapy and below, lasted for a long time, after one single dose, but was reversible upon removal of glucocorticoids. Two nonsteroidal alternative anti-emetic agents did not counteract anticancer treatment and may be sufficient to replace glucocorticoids in cotreatment of carcinoma patients. CONCLUSION: These data demonstrate the need for prospective clinical studies as well as for detailed mechanistic studies of GC-induced cell-type specific pro- and anti-apoptotic signalling.

Glucocorticoid-mediated inhibition of chemotherapy in ovarian carcinomas.

The glucocorticoid dexamethasone is frequently used as a co-treatment in cytotoxic cancer therapy, e.g. to prevent nausea, to protect normal tissue or for other reasons. While the potent pro-apoptotic properties and supportive effects of glucocorticoids to tumour therapy in lymphoid cells are well studied, the impact on the cytotoxic treatment of ovarian carcinoma is unknown. We tested apoptosis-induction, viability, tumour growth and protein expression using established cell lines, primary cell lines freshly isolated from patient material and a xenograft on nude mice. We found a general induction of resistance toward cytotoxic therapy by DEX-co-treatment in most of the examined ovarian cancer cells treated in vitro, ex vivo or in vivo. Resistance occurred independently of cell density and was found at peak plasma levels of dexamethasone and below. Mechanistically, the dexamethasone-induced expression of survival genes may be involved in the resistance. These data show that glucocorticoid-induced resistance is common in ovarian carcinomas implicating that the use of glucocorticoids may be harmful for cancer patients.

L1 on ovarian carcinoma cells is a binding partner for Neuropilin-1 on mesothelial cells.

The progression of ovarian cancer is driven by a variety of cellular factors that are incompletely understood. Binding of tumor cells to normal cells and to soluble factors influence tumor growth, angiogenesis and the stimulation of vascular permeability leading to ascites production. L1 adhesion molecule is overexpressed in ovarian carcinoma and is associated with bad prognosis. One receptor for L1 is Neuropilin-1 (NRP-1) that is also known as a receptor for VEGF(165). In the nervous system a complex of NRP-1 and L1 transmits signals by the neurorepellant Sem3A that is critical for the control of neurite outgrowth. NRP-1 has also been detected in human carcinomas but its function remains unknown. Here, we have examined NRP-1 expression in ovarian carcinoma cell lines and tissue. We report that little NRP-1 protein was detected in primary ovarian carcinoma tissues or established cell lines although mRNA for soluble and transmembrane NRP-1 were detected by RT-PCR. Instead, we observed strong expression of NRP-1 in mesothelial cells, which form the lining of the peritoneum. NRP-1 could serve as an isolation marker for primary mesothelial cells present in ascites fluid. We demonstrate that ovarian cancer cells expressing L1 can bind to NRP-1 overexpressing cells and mesothelial cells. Likewise, soluble L1 isolated from ascites of patients or produced as a fusion protein could bind to NRP-1 overexpressing cells and a direct interaction was demonstrated at the protein level. These findings suggest that L1 can support the binding of ovarian carcinoma cells to mesothelial cells via NRP-1. The L1-NRP-1 binding pathway could contribute to the growth of ovarian carcinomas and to reciprocal signalling between mesothelial cells and tumors.

L1 augments cell migration and tumor growth but not beta3 integrin expression in ovarian carcinomas.

L1 is a neural cell adhesion molecule involved in cell migration, axon growth and guidance. Recent data have shown that L1 is overexpressed in ovarian and endometrial tumors and is associated with bad prognosis. How L1 promotes tumor progression is presently unknown. Here we show that L1 expression is predominantly confined to the invasive front of ovarian carcinomas. Overexpression of L1 in carcinoma cell lines by adenovirus-mediated gene transfer enhanced the haptotactic cell migration on extracellular matrix proteins. Expression of L1 augmented tumor growth of carcinomas xenografted in nonobese diabetic/severe combined immunodeficient mice (NOD/SCID). A recent report has demonstrated L1-dependent upregulation of beta3 integrin involving activation of the extracellular signal-regulated kinase (erk) pathway. We find that L1 and beta3 integrin are not coexpressed in ovarian carcinoma tissues. Overexpression of L1 did not upregulate beta3 integrin in ovarian carcinoma cell lines but could do so in HEK293 cells. Our results suggest that L1 could drive progression by enhancing cell migration and tumor growth but that L1 dependent and erk-regulated gene expression requires cell-type specific elements.