High-level production of wild-type and oxidation-resistant recombinant alpha-1-antitrypsin in glycoengineered CHO cells.

Alpha-1-antitrypsin (A1AT) is a serine protease inhibitor which blocks the activity of serum proteases including neutrophil elastase to protect the lungs. Its deficiency is known to increase the risk of pulmonary emphysema as well as chronic obstructive pulmonary disease. Currently, the only treatment for patients with A1AT deficiency is weekly injection of plasma-purified A1AT. There is still today no commercial source of therapeutic recombinant A1AT, likely due to significant differences in expression host-specific glycosylation profile and/or high costs associated with the huge therapeutic dose needed. Accordingly, we aimed to produce high levels of recombinant wild-type A1AT, as well as a mutated protein (mutein) version for increased oxidation resistance, with N-glycans analogous to human plasma-derived A1AT. To achieve this, we disrupted two endogenous glycosyltransferase genes controlling core α-1,6-fucosylation (Fut8) and α-2,3-sialylation (ST3Gal4) in CHO cells using CRISPR/Cas9 technology, followed by overexpression of human α-2,6-sialyltransferase (ST6Gal1) using a cumate-inducible expression system. Volumetric A1AT productivity obtained from stable CHO pools was 2.5- to 6.5-fold higher with the cumate-inducible CR5 promoter compared to five strong constitutive promoters. Using the CR5 promoter, glycoengineered stable CHO pools were able to produce over 2.1 and 2.8 g/L of wild-type and mutein forms of A1AT, respectively, with N-glycans analogous to the plasma-derived clinical product Prolastin-C. Supplementation of N-acetylmannosamine to the cell culture media during production increased the overall sialylation of A1AT as well as the proportion of bi-antennary and disialylated A2G2S2 N-glycans. These purified recombinant A1AT proteins showed in vitro inhibitory activity equivalent to Prolastin-C and substitution of methionine residues 351 and 358 with valines rendered A1AT significantly more resistant to oxidation. The recombinant A1AT mutein bearing an improved oxidation resistance described in this study could represent a viable biobetter drug, offering a safe and more stable alternative for augmentation therapy.

TNF receptor-associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation.

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

Proteome Profiling of Brain Vessels in a Mouse Model of Cerebrovascular Pathology.

Cerebrovascular pathology that involves altered protein levels (or signaling) of the transforming growth factor beta (TGFß) family has been associated with various forms of age-related dementias, including Alzheimer disease (AD) and vascular cognitive impairment and dementia (VCID). Transgenic mice overexpressing TGFß1 in the brain (TGF mice) recapitulate VCID-associated cerebrovascular pathology and develop cognitive deficits in old age or when submitted to comorbid cardiovascular risk factors for dementia. We characterized the cerebrovascular proteome of TGF mice using mass spectrometry (MS)-based quantitative proteomics. Cerebral arteries were surgically removed from 6-month-old-TGF and wild-type mice, and proteins were extracted and analyzed by gel-free nanoLC-MS/MS. We identified 3602 proteins in brain vessels, with 20 demonstrating significantly altered levels in TGF mice. For total and/or differentially expressed proteins (p ≤ 0.01, ≥ 2-fold change), using multiple databases, we (a) performed protein characterization, (b) demonstrated the presence of their RNA transcripts in both mouse and human cerebrovascular cells, and (c) demonstrated that several of these proteins were present in human extracellular vesicles (EVs) circulating in blood. Finally, using human plasma, we demonstrated the presence of several of these proteins in plasma and plasma EVs. Dysregulated proteins point to perturbed brain vessel vasomotricity, remodeling, and inflammation. Given that blood-isolated EVs are novel, attractive, and a minimally invasive biomarker discovery platform for age-related dementias, several proteins identified in this study can potentially serve as VCID markers in humans.

Mouse embryonic stem cell-derived blood-brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis.

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability. We have developed a mouse BBB model, composed of mouse embryonic stem cell (mESC-D3)-derived brain endothelial-like cells (mBECs), employing a directed monolayer differentiation strategy. Although the mBECs showed a mixed endothelial-epithelial phenotype, they exhibited high transendothelial electrical resistance, inducible by retinoic acid treatment up to 400 Ω cm2. This tight cell barrier resulted in restricted sodium fluorescein permeability (1.7 × 10-5 cm/min), significantly lower than that of bEnd.3 cells (1.02 × 10-3 cm/min) and comparable to human induced pluripotent stem cell (iPSC)-derived BECs (2.0 × 10-5 cm/min). The mBECs expressed tight junction proteins, polarized and functional P-gp efflux transporter and receptor mediated transcytosis (RMT) receptors; collectively important criteria for studying barrier regulation and drug delivery applications in the CNS. In this study, we compared transport of a panel of antibodies binding species selective or cross-reactive epitopes on BBB RMT receptors in both the mBEC and human iPSC-derived BEC model, to demonstrate discrimination of species-specific BBB transport mechanisms.

Simplifying glycan monitoring of complex antigens such as the SARS-CoV-2 spike to accelerate vaccine development.

Glycosylation is a key quality attribute that must be closely monitored for protein therapeutics. Established assays such as HILIC-Fld of released glycans and LC-MS of glycopeptides work well for glycoproteins with a few glycosylation sites but are less amenable for those with multiple glycosylation sites, resulting in complex datasets that are time consuming to generate and difficult to analyze. As part of efforts to improve preparedness for future pandemics, researchers are currently assessing where time can be saved in the vaccine development and production process. In this context, we evaluated if neutral and acidic monosaccharides analysis via HPAEC-PAD could be used as a rapid and robust alternative to LC-MS and HILIC-Fld for monitoring glycosylation between protein production batches. Using glycoengineered spike proteins we show that the HPAEC-PAD monosaccharide assays could quickly and reproducibly detect both major and minor glycosylation differences between batches. Moreover, the monosaccharide results aligned well with those obtained by HILIC-Fld and LC-MS.

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells.

BACKGROUND: As the COVID-19 pandemic continues to evolve, novel vaccines need to be developed that are readily manufacturable and provide clinical efficacy against emerging SARS-CoV-2 variants. Virus-like particles (VLPs) presenting the spike antigen at their surface offer remarkable benefits over other vaccine antigen formats; however, current SARS-CoV-2 VLP vaccines candidates in clinical development suffer from challenges including low volumetric productivity, poor spike antigen density, expression platform-driven divergent protein glycosylation and complex upstream/downstream processing requirements. Despite their extensive use for therapeutic protein manufacturing and proven ability to produce enveloped VLPs, Chinese Hamster Ovary (CHO) cells are rarely used for the commercial production of VLP-based vaccines. METHODS: Using CHO cells, we aimed to produce VLPs displaying the full-length SARS-CoV-2 spike. Affinity chromatography was used to capture VLPs released in the culture medium from engineered CHO cells expressing spike. The structure, protein content, and glycosylation of spikes in VLPs were characterized by several biochemical and biophysical methods. In vivo, the generation of neutralizing antibodies and protection against SARS-CoV-2 infection was tested in mouse and hamster models. RESULTS: We demonstrate that spike overexpression in CHO cells is sufficient by itself to generate high VLP titers. These VLPs are evocative of the native virus but with at least three-fold higher spike density. In vivo, purified VLPs elicit strong humoral and cellular immunity at nanogram dose levels which grant protection against SARS-CoV-2 infection. CONCLUSIONS: Our results show that CHO cells are amenable to efficient manufacturing of high titers of a potently immunogenic spike protein-based VLP vaccine antigen.

Virus-like particles (VLPs) have a structure that is similar to viruses but they cannot cause infection or illness. If VLPs are injected into the body they produce an immune response similar to that seen following infection by a virus. This means that VLPs can be used as vaccines against viruses that cause illness in people. Many drugs, named biologics, are manufactured using living cells, including cells that were originally derived from Chinese Hamster Ovaries (CHO cells). We developed a simple method to produce VLPs similar to the SARS-CoV-2 virus in CHO cells. We show that vaccination of rodents with these VLPs prevents them from becoming ill following infection with SARS-CoV-2. These VLPs could become a part of an alternative, easily produced vaccine for the prevention of COVID-19 in humans.

Characterization of Extracellular Vesicles Secreted in Lentiviral Producing HEK293SF Cell Cultures.

Lentiviral vectors (LVs) are a powerful tool for gene and cell therapy and human embryonic kidney cells (HEK293) have been extensively used as a platform for production of these vectors. Like most cells and cellular tissues, HEK293 cells release extracellular vesicles (EVs). EVs released by cells share similar size, biophysical characteristics and even a biogenesis pathway with cell-produced enveloped viruses, making it a challenge to efficiently separate EVs from LVs. Thus, EVs co-purified with LVs during downstream processing, are considered "impurities" in the context of gene and cell therapy. A greater understanding of EVs co-purifying with LVs is needed to enable improved downstream processing. To that end, EVs from an inducible lentivirus producing cell line were studied under two conditions: non-induced and induced. EVs were identified in both conditions, with their presence confirmed by transmission electron microscopy and Western blot. EV cargos from each condition were then further characterized by a multi-omic approach. Nineteen proteins were identified by mass spectrometry as potential EV markers to differentiate EVs in LV preparations. Lipid composition of EV preparations before and after LV induction showed similar enrichment in phosphatidylserine. RNA cargos in EVs showed enrichment in transcripts involved in viral processes and binding functions. These findings provide insights on the product profile of lentiviral preparations and could support the development of improved separation strategies aimed at removing co-produced EVs.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human.

Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.

A charge-suppressing strategy for probing protein methylation.

Methylation of arginine and lysine (RK) residues play essential roles in epigenetics and the regulation of gene expression. However, research in this area is often hindered by the lack of effective tools for probing the protein methylation. Here, we present an antibody-free strategy to capture protein methylation on RK residues by using chemical reactions to eliminate the charges on un-modified RK residues and peptide N-termini. Peptides containing methylated RK residues remain positively charged and are then enriched by strong cation exchange chromatography, followed by high-resolution mass spectrometry identification.