Transcriptomics and cell painting analysis reveals molecular and morphological features associated with fed-batch production performance in CHO recombinant clones.

Stable, highly productive mammalian cells are critical for manufacturing affordable and effective biological medicines. Establishing a rational design of optimal biotherapeutic expression systems requires understanding how cells support the high demand for efficient biologics production. To that end, we performed transcriptomics and high-throughput imaging studies to identify putative genes and morphological features that underpin differences in antibody productivity among clones from a Chinese hamster ovary cell line. During log phase growth, we found that the expression of genes involved in biological processes related to cellular morphology varied significantly between clones with high specific productivity (qP > 35 pg/cell/day) and low specific productivity (qP < 20 pg/cell/day). At Day 10 of a fed-batch production run, near peak viable cell density, differences in gene expression related to metabolism, epigenetic regulation, and proliferation became prominent. Furthermore, we identified a subset of genes whose expression predicted overall productivity, including glutathione synthetase (Gss) and lactate dehydrogenase A (LDHA). Finally, we demonstrated the feasibility of cell painting coupled with high-throughput imaging to assess the morphological properties of intracellular organelles in relation to growth and productivity in fed-batch production. Our efforts lay the groundwork for systematic elucidation of clone performance using a multiomics approach that can guide future process design strategies.

Significant impact of mTORC1 and ATF4 pathways in CHO cell recombinant protein production induced by CDK4/6 inhibitor.

The CDK4/6 inhibitor has been shown to increase recombinant protein productivity in Chinese hamster ovary (CHO) cells. Therefore, we investigated the mechanism that couples cell-cycle inhibitor (CCI) treatment with protein productivity utilizing proteomics and phosphoproteomics. We identified mTORC1 as a critical early signaling event that preceded boosted productivity. Following CCI treatment, mTOR exhibited a transient increase in phosphorylation at a novel site that is also conserved in humans and mouse. Upstream of mTORC1, increased phosphorylation of AKT1S1 and decreased phosphorylation of RB1 may provide molecular links between CDK4/6 inhibition and mTORC1. Downstream, increased EIF4EBP1 phosphorylation was observed, which can mediate cap-dependent translation. In addition, the collective effect of increased phosphorylation of RPS6, increased phosphorylation of regulators of RNA polymerase I, and increased protein expression in the transfer RNA-aminoacylation pathway may contribute to enhancing the translational apparatus for increased productivity. In concert, an elevated stress response via GCN2/EIF2AK4-ATF4 axis persisted over the treatment course, which may link mTOR to downstream responses including the unfolded protein response and autophagy to enhance proper protein folding and secretion. Together, this comprehensive proteomics and phosphoproteomics characterization of CCI-treated CHO cells offers insights into understanding multiple aspects of signaling events resulting from CDK4/CDK6 inhibition.

Differential effects on natural killer cell production by membrane-bound cytokine stimulations.

Robust manufacturing production of natural killer (NK) cells has been challenging in allogeneic NK cell-based therapy. Here, we compared the impact of cytokines on NK cell expansion by developing recombinant K562 feeder cell lines expressing membrane-bound cytokines, mIL15, mIL21, and 41BBL, individually or in combination. We found that 41BBL played a dominant role in promoting up to 500,000-fold of NK cell expansion after a 21-day culture process without inducing exhaustion. However, 41BBL stimulation reduced the overall cytotoxic activity of NK cells when combined with mIL15 and/or mIL21. Additionally, long-term stimulation with mIL15 and/or mIL21, but not 41BBL, increased CD56 expression and the CD56bright population, which is unexpectedly correlated with NK cell cytotoxicity. By conducting single-cell sequencing, we identified distinct subpopulations of NK cells induced by different cytokines, including an adaptive-like CD56bright CD16- CD49a+ subset induced by mIL15. Through gene expression analysis, we found that different cytokines modulated signaling pathways and target genes involved in cell cycle, senescence, self-renewal, migration, and maturation in different ways. Together, our study demonstrates that cytokine signaling pathways play distinct roles in NK cell expansion and differentiation, which sheds light on NK cell process designs to improve productivity and product quality.

Epigenetic comparison of CHO hosts and clones reveals divergent methylation and transcription patterns across lineages.

In this study, we examined DNA methylation and transcription profiles of recombinant clones derived from two different Chinese hamster ovary hosts. We found striking epigenetic differences between the clones, with global hypomethylation in the host 1 clones that produce bispecific antibody with higher productivity and complex assembly efficiency. Whereas the methylation patterns were found mostly inherited from the host, the host 1 clones exhibited continued demethylation reflected by the hypomethylation of newly emerged differential methylation regions (DMRs) even at the clone development stage. Several interconnected biological functions and pathways including cell adhesion, regulation of ion transport, and cholesterol biosynthesis were significantly altered between the clones at the RNA expression level and contained DMR in the promoter and/or gene-body of the transcripts, suggesting epigenetic regulation. Indeed, expression changes of epigenetic regulators were observed including writers (Dnmt1, Setdb1), readers (Mecp2), and erasers (Tet3, Kdm3a, Kdm1b/5c) involved in CpG methylation, histone methylation, and heterochromatin maintenance. In addition, we identified putative transcription factors that may be readers or effectors of the epigenetic regulation in these clones. By combining transcriptomics with DNA methylation data, we identified potential processes and factors that may contribute to the variability in cell physiology between different production hosts.

Rhinovirus-16 induced temporal interferon responses in nasal epithelium links with viral clearance and symptoms.

BACKGROUND: The temporal in vivo response of epithelial cells to a viral challenge and its association with viral clearance and clinical outcomes has been largely unexplored in asthma. OBJECTIVE: To determine gene expression profiles over time in nasal epithelial cells (NECs) challenged in vivo with rhinovirus-16 (RV16) and compare to nasal symptoms and viral clearance. METHODS: Patients with stable mild to moderate asthma (n = 20) were challenged intranasally with RV16. Nasal brush samples for RNA sequencing were taken 7 days prior to infection and 3, 6 and 14 days post-infection, and blood samples 4 days prior to infection and day 6 post-infection. Viral load was measured in nasal lavage fluid at day 3, 6 and 14. RESULTS: Top differentially (>2.5-fold increase) expressed gene sets in NECs post-RV16 at days 3 and 6, compared with baseline, were interferon alpha and gamma response genes. Patients clearing the virus within 6 days (early resolvers) had a significantly increased interferon response at day 6, whereas those having cleared the virus by day 14 (late resolvers) had significantly increased responses at day 3, 6 and 14. Interestingly, patients not having cleared the virus by day 14 (non-resolvers) had no enhanced interferon responses at any of these days. The daily Cold Symptom Scores (CSS) peaked at days 3 to 5 and correlated positively with interferon response genes at day 3 (R = 0.48), but not at other time-points. Interferon response genes were also enhanced in blood at day 6 after RV16 challenge. CONCLUSION AND CLINICAL RELEVANCE: This study shows that viral load and clearance varies markedly over time in mild to moderate asthma patients exposed to a fixed RV16 dose. The host's nasal interferon response to RV16 at day 3 is associated with upper respiratory tract symptoms. The temporal interferon response in nasal epithelium associates with viral clearance in the nasal compartment.

An In-Vitro Study of the Expansion and Transcriptomics of CD4+ and CD8+ Naïve and Memory T Cells Stimulated by IL-2, IL-7 and IL-15.

The growth of T cells ex vivo for the purpose of T cell therapies is a rate-limiting step in the overall process for cancer patients to achieve remission. Growing T cells is a fiscally-, time-, and resource-intensive process. Cytokines have been shown to accelerate the growth of T cells, specifically IL-2, IL-7, and IL-15. Here a design of experiments was conducted to optimize the growth rate of different naïve and memory T cell subsets using combinations of cytokines. Mathematical models were developed to study the impact of IL-2, IL-7, and IL-15 on the growth of T cells. The results show that CD4+ and CD8+ naïve T cells grew effectively using moderate IL-2 and IL-7 in combination, and IL-7, respectively. CD4+ and CD8+ memory cells favored moderate IL-2 and IL-15 in combination and moderate IL-7 and IL-15 in combination, respectively. A statistically significant interaction was observed between IL-2 and IL-7 in the growth data of CD4+ naïve T cells, while the interaction between IL-7 and IL-15 was found for CD8+ naïve T cells. The important genes and related signaling pathways and metabolic reactions were identified from the RNA sequencing data for each of the four subsets stimulated by each of the three cytokines. This systematic investigation lays the groundwork for studying other T cell subsets.

Chromosomal instability drives convergent and divergent evolution toward advantageous inherited traits in mammalian CHO bioproduction lineages.

Genetic instability of Chinese hamster ovary (CHO) cells is implicated in production inconsistency through poorly defined mechanisms. Using a multi-omics approach, we analyzed the variations of CHO lineages derived from CHO-K1 cells. We identify an equilibrium between random genetic variation of the CHO genome and heritable traits driven by culture conditions, selection criteria, and genetic linkage. These inherited changes are associated with the selection pressures related to serum removal, suspension culture transition, protein expression, and secretion. We observed that a haploid reduction of a Chromosome 2 region after serum-free, suspension adaptation, was consistently inherited, suggesting common adaptation mechanisms. Genetic variations also included ∼200 insertions/deletions, ∼1000 single-nucleotide polymorphisms, and ∼300-2000 copy number variations, which were exacerbated after gene editing. In addition, heterochromatic chromosomes were preferentially lost as cells continuously evolved. Together, these observations demonstrate a highly plastic signature for adapted CHO cells and paves the way towards future host cell engineering.

Detection of respiratory syncytial virus defective genomes in nasal secretions is associated with distinct clinical outcomes.

Respiratory syncytial virus (RSV) causes respiratory illness in children, immunosuppressed individuals and the elderly. However, the viral factors influencing the clinical outcome of RSV infections remain poorly defined. Defective viral genomes (DVGs) can suppress virus replication by competing for viral proteins and by stimulating antiviral immunity. We studied the association between detection of DVGs of the copy-back type and disease severity in three RSV A-confirmed cohorts. In hospitalized children, detection of DVGs in respiratory samples at or around the time of admission associated strongly with more severe disease, higher viral load and a stronger pro-inflammatory response. Interestingly, in experimentally infected adults, the presence of DVGs in respiratory secretions differentially associated with RSV disease severity depending on when DVGs were detected. Detection of DVGs early after infection associated with low viral loads and mild disease, whereas detection of DVGs late after infection, especially if DVGs were present for prolonged periods, associated with high viral loads and severe disease. Taken together, we demonstrate that the kinetics of DVG accumulation and duration could predict clinical outcome of RSV A infection in humans, and thus could be used as a prognostic tool to identify patients at risk of worse clinical disease.

Evaluation of phenoxybenzamine in the CFA model of pain following gene expression studies and connectivity mapping.

BACKGROUND: We have previously used the rat 4 day Complete Freund's Adjuvant (CFA) model to screen compounds with potential to reduce osteoarthritic pain. The aim of this study was to identify genes altered in this model of osteoarthritic pain and use this information to infer analgesic potential of compounds based on their own gene expression profiles using the Connectivity Map approach. RESULTS: Using microarrays, we identified differentially expressed genes in L4 and L5 dorsal root ganglia (DRG) from rats that had received intraplantar CFA for 4 days compared to matched, untreated control animals. Analysis of these data indicated that the two groups were distinguishable by differences in genes important in immune responses, nerve growth and regeneration. This list of differentially expressed genes defined a "CFA signature". We used the Connectivity Map approach to identify pharmacologic agents in the Broad Institute Build02 database that had gene expression signatures that were inversely related ('negatively connected') with our CFA signature. To test the predictive nature of the Connectivity Map methodology, we tested phenoxybenzamine (an alpha adrenergic receptor antagonist) - one of the most negatively connected compounds identified in this database - for analgesic activity in the CFA model. Our results indicate that at 10 mg/kg, phenoxybenzamine demonstrated analgesia comparable to that of Naproxen in this model. CONCLUSION: Evaluation of phenoxybenzamine-induced analgesia in the current study lends support to the utility of the Connectivity Map approach for identifying compounds with analgesic properties in the CFA model.

Neutrophilic inflammation in the respiratory mucosa predisposes to RSV infection.

The variable outcome of viral exposure is only partially explained by known factors. We administered respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil activation before exposure was highly predictive of symptomatic RSV disease. This was associated with a rapid, presymptomatic decline in mucosal interleukin-17A (IL-17A) and other mediators. Conversely, those who resisted infection showed presymptomatic activation of IL-17- and tumor necrosis factor-related pathways. Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by preinfection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease mediated by pulmonary CD8+ T cell infiltration. Thus, mucosal neutrophilic inflammation at the time of RSV exposure enhances susceptibility, revealing dynamic, time-dependent local immune responses before symptom onset and explaining the as-yet unpredictable outcomes of pathogen exposure.

Live Cell Membranome cDNA Screen: A Novel Homogenous Live Cell Binding Assay to Study Membrane Protein-Ligand Interaction.

Interactions between transmembrane receptors and their ligands play important roles in normal biological processes and pathological conditions. However, the binding partners for many transmembrane-like proteins remain elusive. To identify potential ligands of these orphan receptors, we developed a screening platform using a homogenous nonwash binding assay in live cells. A collection of ~1900 cDNA clones, encoding full-length membrane proteins, was assembled. As a proof of concept, cDNA clones were individually transfected into CHO-K1 cells in a high-throughput format, and soluble PD-L1-Fc fusion protein was used as bait. The interaction between the putative receptor and PD-L1-Fc was then detected by Alexa Fluor 647 conjugated anti-human immunoglobulin G Fc antibody and visualized using the Mirrorball fluorescence plate cytometer. As expected, PDCD1, the gene encoding programmed cell death protein 1 (PD-1), was revealed as the predominant hit. In addition, three genes that encode Fc receptors (FCGR1A, FCGR1B, and FCGR2A) were also identified as screen hits as the result of the Fc-tag fused to PD-L1, which has provided a reliable internal control for the screen. Furthermore, the potential of using a biotinylated ligand was explored and established to expand the versatility of the cDNA platform. This novel screening platform not only provides a powerful tool for the identification of ligands for orphan receptors but also has the potential for small-molecule target deconvolution.

Developing timely insights into comparative effectiveness research with a text-mining pipeline.

Comparative effectiveness research (CER) provides evidence for the relative effectiveness and risks of different treatment options and informs decisions made by healthcare providers, payers, and pharmaceutical companies. CER data come from retrospective analyses as well as prospective clinical trials. Here, we describe the development of a text-mining pipeline based on natural language processing (NLP) that extracts key information from three different trial data sources: NIH ClinicalTrials.gov, WHO International Clinical Trials Registry Platform (ICTRP), and Citeline Trialtrove. The pipeline leverages tailored terminologies to produce an integrated and structured output, capturing any trials in which pharmaceutical products of interest are compared with another therapy. The timely information alerts generated by this system provide the earliest and most complete picture of emerging clinical research.

Application of an automated natural language processing (NLP) workflow to enable federated search of external biomedical content in drug discovery and development.

External content sources such as MEDLINE(®), National Institutes of Health (NIH) grants and conference websites provide access to the latest breaking biomedical information, which can inform pharmaceutical and biotechnology company pipeline decisions. The value of the sites for industry, however, is limited by the use of the public internet, the limited synonyms, the rarity of batch searching capability and the disconnected nature of the sites. Fortunately, many sites now offer their content for download and we have developed an automated internal workflow that uses text mining and tailored ontologies for programmatic search and knowledge extraction. We believe such an efficient and secure approach provides a competitive advantage to companies needing access to the latest information for a range of use cases and complements manually curated commercial sources.

Diminished proliferation of B blast cell in response to cytokines in ethanol-consuming mice.

We and others have demonstrated that ethanol suppresses the antibody response in humans and animals. The purpose of this study was to determine whether ethanol affects cytokine-induced proliferative responses of splenic B blast cells, and whether the decreased response was due to an imbalance of the cytokine activity. Thus, the ability of spleen cells from individual ethanol-diet-fed C57BL/6 mice to proliferate and produce cytokines was determined. The ability of anti-IgM monoclonal antibodies (mAb)-activated splenic B blast cells in response to mouse recombinant IL-2 (rIL-2) or rIL-4 was also assessed. A thymidine incorporation assay was used to determine cell proliferation, and the conventional bioassays for cytokine-dependent cell proliferation were used for determining the bioactivity of cytokines. Data were analyzed with general linear model procedure. Our results showed that ethanol weakened the proliferative response of B cells in response to mitogen as well as to mouse rIL-2 and rIL-4. The decreased B cell responses may result from an increase in the production of IL-4 by helper T cells. Finally, in the presence of excessive dose of rIL-4, the proliferative responses of B blast cells from all three groups of mice were diminished (p<0.01). Thus, our data clearly indicated that the diminished B cell proliferation in ethanol-consuming mice was due in part to an excessive amount of IL-4 produced and an inability of the B cell to interact properly with IL-4 that was secreted by helper T cells. The results should extend our basic understanding of the mechanism by which chronic alcoholism impairs the interactions and interdependence of T- and B-cell immune functions.

The yeast pafl-rNA polymerase II complex is required for full expression of a subset of cell cycle-regulated genes.

We have previously described an alternative form of RNA polymerase II in yeast lacking the Srb and Med proteins but including Pafl, Cdc73, Hprl, and Ccr4. The Pafl-RNA polymerase II complex (Paf1 complex) acts in the same pathway as the Pkc1-mitogen-activated protein kinase cascade and is required for full expression of many cell wall biosynthetic genes. The expression of several of these cell integrity genes, as well as many other Paf1-requiring genes identified by differential display and microarray analyses, is regulated during the cell cycle. To determine whether the Paf1 complex is required for basal or cyclic expression of these genes, we assayed transcript abundance throughout the cell cycle. We found that transcript abundance for a subset of cell cycle-regulated genes, including CLN1, HO, RNR1, and FAR1, is reduced from 2- to 13-fold in a paf1delta strain, but that this reduction is not promoter dependent. Despite the decreased expression levels, cyclic expression is still observed. We also examined the possibility that the Paf1 complex acts in the same pathway as either SBF (Swi4/Swi6) or MBF (Mbp1/Swi6), the partially redundant cell cycle transcription factors. Consistent with the possibility that they have overlapping essential functions, we found that loss of Paf1 is lethal in combination with loss of Swi4 or Swi6. In addition, overexpression of either Swi4 or Mbp1 suppresses some paf1delta phenotypes. These data establish that the Paf1 complex plays an important role in the essential regulatory pathway controlled by SBF and MBF.