Mechanisms underlying epigenetic and transcriptional heterogeneity in Chinese hamster ovary (CHO) cell lines.

BACKGROUND: Recombinant cell lines developed for therapeutic antibody production often suffer instability or lose recombinant protein expression during long-term culture. Heterogeneous gene expression among cell line subclones may result from epigenetic modifications of DNA or histones, the protein component of chromatin. We thus investigated in such cell lines, DNA methylation and the chromatin environment along the human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter in an antibody protein-expression vector which was integrated into the Chinese hamster ovary (CHO) cell line genome. RESULTS: We analyzed four PT1-CHO cell lines which exhibited losses of protein expression at advanced passage number (>P35) growing in adherent conditions and in culture medium with 10 % FCS. These cell lines exhibited different integration sites and transgene copy numbers as determined by fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR), respectively. By qRT-PCR, we analyzed the recombinant mRNA expression and correlated it with DNA methylation and with results from various approaches interrogating the chromatin landscape along the EEF1A1 promoter region. Each PT1-CHO cell line displayed specific epigenetic signatures or chromatin marks correlating with recombinant mRNA expression. The cell line with the lowest recombinant mRNA expression (PT1-1) was characterized by the highest nucleosome occupancy and displayed the lowest enrichment for histone marks associated with active transcription. In contrast, the cell line with the highest recombinant mRNA expression (PT1-55) exhibited the highest numbers of formaldehyde-assisted isolation of regulatory elements (FAIRE)-enriched regions, and was marked by enrichment for histone modifications H3K9ac and H3K9me3. Another cell line with the second highest recombinant mRNA transcription and the most stable protein expression (PT1-7) had the highest enrichments of the histone variants H3.3 and H2A.Z, and the histone modification H3K9ac. A further cell line (PT1-30) scored the highest enrichments for the bivalent marks H3K4me3 and H3K27me3. Finally, DNA methylation made a contribution, but only in the culture medium with reduced FCS or in a different expression vector. CONCLUSIONS: Our results suggest that the chromatin state along the EEF1A1 promoter region can help predict recombinant mRNA expression, and thus may assist in selecting desirable clones during cell line development for protein production.

Repetitive Exposure to Bacteriophage Cocktails against Pseudomonas aeruginosa or Escherichia coli Provokes Marginal Humoral Immunity in Naïve Mice.

Phage therapy of ventilator-associated pneumonia (VAP) is of great interest due to the rising incidence of multidrug-resistant bacterial pathogens. However, natural or therapy-induced immunity against therapeutic phages remains a potential concern. In this study, we investigated the innate and adaptive immune responses to two different phage cocktails targeting either Pseudomonas aeruginosa or Escherichia coli-two VAP-associated pathogens-in naïve mice without the confounding effects of a bacterial infection. Active or UV-inactivated phage cocktails or buffers were injected intraperitoneally daily for 7 days in C57BL/6J wild-type mice. Blood cell analysis, flow cytometry analysis, assessment of phage distribution and histopathological analysis of spleens were performed at 6 h, 10 days and 21 days after treatment start. Phages reached the lungs and although the phage cocktails were slightly immunogenic, phage injections were well tolerated without obvious adverse effects. No signs of activation of innate or adaptive immune cells were observed; however, both active phage cocktails elicited a minimal humoral response with secretion of phage-specific antibodies. Our findings show that even repetitive injections lead only to a minimal innate and adaptive immune response in naïve mice and suggest that systemic phage treatment is thus potentially suitable for treating bacterial lung infections.

Phage Therapy in Germany-Update 2023.

Bacteriophage therapy holds promise in addressing the antibiotic-resistance crisis, globally and in Germany. Here, we provide an overview of the current situation (2023) of applied phage therapy and supporting research in Germany. The authors, an interdisciplinary group working on patient-focused bacteriophage research, addressed phage production, phage banks, susceptibility testing, clinical application, ongoing translational research, the regulatory situation, and the network structure in Germany. They identified critical shortcomings including the lack of clinical trials, a paucity of appropriate regulation and a shortage of phages for clinical use. Phage therapy is currently being applied to a limited number of patients as individual treatment trials. There is presently only one site in Germany for large-scale good-manufacturing-practice (GMP) phage production, and one clinic carrying out permission-free production of medicinal products. Several phage banks exist, but due to varying institutional policies, exchange among them is limited. The number of phage research projects has remarkably increased in recent years, some of which are part of structured networks. There is a demand for the expansion of production capacities with defined quality standards, a structured registry of all treated patients and clear therapeutic guidelines. Furthermore, the medical field is still poorly informed about phage therapy. The current status of non-approval, however, may also be regarded as advantageous, as insufficiently restricted use of phage therapy without adequate scientific evidence for effectiveness and safety must be prevented. In close coordination with the regulatory authorities, it seems sensible to first allow some centers to treat patients following the Belgian model. There is an urgent need for targeted networking and funding, particularly of translational research, to help advance the clinical application of phages.

A novel ACE2 decoy for both neutralization of SARS-CoV-2 variants and killing of infected cells.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to millions of infections and deaths worldwide. As this virus evolves rapidly, there is a high need for treatment options that can win the race against new emerging variants of concern. Here, we describe a novel immunotherapeutic drug based on the SARS-CoV-2 entry receptor ACE2 and provide experimental evidence that it cannot only be used for (i) neutralization of SARS-CoV-2 in vitro and in SARS-CoV-2-infected animal models but also for (ii) clearance of virus-infected cells. For the latter purpose, we equipped the ACE2 decoy with an epitope tag. Thereby, we converted it to an adapter molecule, which we successfully applied in the modular platforms UniMAB and UniCAR for retargeting of either unmodified or universal chimeric antigen receptor-modified immune effector cells. Our results pave the way for a clinical application of this novel ACE2 decoy, which will clearly improve COVID-19 treatment.

Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection.

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.

On the structural diversity of Shiga toxin glycosphingolipid receptors in lymphoid and myeloid cells determined by nanoelectrospray ionization tandem mass spectrometry.

Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galalpha4Galbeta4Glcbeta1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS(1) and MS(2) analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells.

Efficiency improvement of an antibody production process by increasing the inoculum density.

Increasing economic pressure is the main driving force to enhance the efficiency of existing processes. We developed a perfusion strategy for a seed train reactor to generate a higher inoculum density for a subsequent fed batch production culture. A higher inoculum density can reduce culture duration without compromising product titers. Hence, a better capacity utilization can be achieved. The perfusion strategy was planned to be implemented in an existing large scale antibody production process. Therefore, facility and process constraints had to be considered. This article describes the initial development steps. Using a proprietary medium and a Chinese hamster ovary cell line expressing an IgG antibody, four different cell retention devices were compared in regard to retention efficiency and reliability. Two devices were selected for further process refinement, a centrifuge and an inclined gravitational settler. A concentrated feed medium was developed to meet facility constraints regarding maximum accumulated perfundate volume. A 2-day batch phase followed by 5 days of perfusion resulted in cell densities of 1.6 × 10(10) cells L(-1) , a 3.5 fold increase compared to batch cultivations. Two reactor volumes of concentrated feed medium were needed to achieve this goal. Eleven cultivations were carried out in bench and 50 L reactors showing acceptable reproducibility and ease of scale up. In addition, it was shown that at least three perfusion phases can be combined within a repeated perfusion strategy.