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Recombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal–knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies.

Cancer is a multifactorial disease that constitutes a serious public health problem worldwide. Prostate cancer advanced stages are associated with the development of androgen-independent tumors and an apoptosis-resistant phenotype that progresses to metastasis. By studying androgen-independent lymphoid nodule carcinoma of the prostate (LNCaP) cells induced to apoptosis by serum elimination, we identified the activation of a non-selective cationic channel of 23pS conductance that promotes incoming Ca2+ currents, as well as apoptosis final stages. arp2cDNA was isolated and identified to be of the same cell type, and mRNA was expressed in Xenopus laevis oocytes, which was found to be associated with the activation of incoming Ca2+ currents and induction to apoptosis. cDNA, which encodes the ARP2 protein, was overexpressed in LNCaP cells and Chinese hamster ovary cells, which induced apoptosis. Our evidence suggests that protein ARP2 overexpression and transit to the cell membrane allows an increased Ca2+ incoming current that initiates the apoptosis process in epithelial-type cells whose phenotype shows resistance to programmed cell death.

Polymeric nanoparticles acting as sources of selenium (Se) are currently an interesting topic in cancer chemotherapy. In this study, polyglycerol dendrimer (DPGLy) was functionalized with seleno-methyl-selenocysteine (SeMeCys) by means of Steglich esterification with 4-dimethylaminopyridine/(l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC/DMAP) and cerium chloride as cocatalyst in acetonitrile at quantitative yields of 98 ± 1%. The SeMeCys coupling DPGLy efficiency vs. time were determined by Fourier Transform infrared spectroscopy (FTIR) and ultraviolet-visible (UV-Vis) spectroscopy. The cytotoxic effects of SeMeCys-DPGLy on the Chinese Hamster ovary cell line (CHO-K1) and head and neck squamous cell carcinoma (HNSCC) cells line were assessed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. No signs of general toxicity of SeMeCys-DPGLy against CHO-K1 cells were detectable at which cell viability was greater than 98%. MTS assays revealed that SeMeCys-DPGLy reduced HNSCC cell viability and proliferation at higher doses and long incubation times.

The zona pellucida (ZP) is an extracellular matrix surrounding mammalian oocytes. It is composed of three to four glycoproteins, ZP1-ZP4. ZP3 is essential for sperm binding and zona matrix formation. Here, we identified a novel heterozygous mutation (NM_001110354.2:c.502_504delGAG) of ZP3, occurring in a pair of sisters with empty follicle syndrome (EFS). A mouse model with the same mutation was established using the CRISPR/Cas9 gene-editing system. As in the above family, F0 -, F1 -, and F2 -generation female mice with the mutation were all infertile. Further analysis using the Chinese hamster ovary cells (CHO-K1) also showed that this mutation weakens the strength of binding between ZP3 and ZP2, which hinders the assembly of ZP and results in unstable ZP formation. Immunohistochemical analysis using ovarian serial sections in both humans and mice demonstrated that the ZP of preantral follicles was thinner than normal control, or even absent. Our study presents a new gene mutation that leads to EFS, providing new evidence and support for the genetic diagnosis of infertile individuals with similar phenotypes. Our results also show that the loop of ZP3 is not only a linker between two amphiphilic helices but may play a critical role in specifying the correct heterodimerization partner. This article is protected by copyright. All rights reserved.

The high molecular weight (HMW) size variants present in therapeutic monoclonal antibody (mAb) samples need to be closely monitored and characterized due to their impact on product safety and efficacy. Because of the complexity and often low abundances in final drug substance (DS) samples, characterization of such HMW species is challenging and traditionally requires offline enrichment of the HMW species followed by analysis using various analytical tools. Here, we report the development of a postcolumn denaturation-assisted native SEC-MS method that allows rapid and in-depth characterization of mAb HMW species directly from unfractionated DS samples. This method not only provides high-confidence identification of HMW complexes based on accurate mass measurement of both the intact assembly and the constituent subunits but also allows in-depth analysis of the interaction nature and location. In addition, using the extracted ion chromatograms, derived from high-quality, native-like mass spectra, the elution profiles of each noncovalent and/or nondissociable complex can be readily reconstructed, facilitating the comprehension of a complex HMW profile. The utility of this novel method was demonstrated in different applications, ranging from enriched HMW characterization at late stage development, comparability assessment due to process changes, and forced degradation study of coformulated mAbs. As this method does not require prior enrichment, it is thus desirable for providing both rapid and in-depth characterization of HMW species during the development of therapeutic mAbs.

CD40 is a potent activating receptor within the TNFR family expressed on APCs of the immune system, and it regulates many aspects of B and T cell immunity via interaction with CD40 ligand (CD40L; CD154) expressed on the surface of activated T cells. Soluble CD40L and agonistic mAbs directed to CD40 are being explored as adjuvants in therapeutic or vaccination settings. Some anti-CD40 Abs can synergize with soluble monomeric CD40L. We show that direct fusion of CD40L to certain agonistic anti-CD40 Abs confers superagonist properties, reducing the dose required for efficacy, notably greatly increasing total cytokine secretion by human dendritic cells. The tetravalent configuration of anti-CD40–CD40L Abs promotes CD40 cell surface clustering and internalization and is the likely mechanism of increased receptor activation. CD40L fused to either the L or H chain C termini, with or without flexible linkers, were all superagonists with greater potency than CD40L trimer. The increased anti-CD40–CD40L Ab potency was independent of higher order aggregation. Moreover, the anti-CD40–CD40L Ab showed higher potency in vivo in human CD40 transgenic mice compared with the parental anti-CD40 Ab. To broaden the concept of fusing agonistic Ab to natural ligand, we fused OX40L to an agonistic OX40 Ab, and this resulted in dramatically increased efficacy for proliferation and cytokine production of activated human CD4+ T cells as well as releasing the Ab from dependency on cross-linking. This work shows that directly fusing antireceptor Abs to ligand is a useful strategy to dramatically increase agonist potency.

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen-binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full-length antibodies are mammalian cell-based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small- and large-scale Fab production. Using a panel of full-length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full-length antibody format resulted in the recovery of fewer mini-pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody-producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full-length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats.

Peptides are notoriously known to display very short in vivo half-lives often measured in minutes which in many cases greatly reduces or eliminates sufficient in vivo efficacy. To obtain long half-lives allowing for up to once-weekly dosing regimen, fatty acid acylation (lipidation) have been used to non-covalently associate the peptide to serum albumin thus serving as a circulating depot. This approach is generally considered in the scientific and patent community as a standard approach to protract almost any given peptide. However, it is not trivial to prolong the half-life of peptides by lipidation and still maintain high potency and good formulation properties. Here we show that attaching a fatty acid to the obesity-drug relevant peptide PYY3-36 is not sufficient for long pharmacokinetics (PK), since the position in the backbone, but also type of fatty acid and linker strongly influences PK and potency. Furthermore, understanding the proteolytic stability of the backbone is key to obtain long half-lives by lipidation, since backbone cleavage still occurs while associated to albumin. Having identified a PYY analogue with a sufficient half-life, we show that in combination with a GLP-1 analogue, liraglutide, additional weight loss can be achieved in the obese minipig model.

The β-amyloid peptide (Aβ) is the main constituent of senile plaques, a typical hallmark of Alzheimer’s disease (AD). Monomeric Aβ is generated through sequential processing of the amyloid precursor protein (APP), with a final step involving γ-secretase activity. In AD, Aβ monomers assemble in oligomers and ultimately fibrils depositing in plaques. Importantly, Aβ toxicity appears related to its soluble oligomeric intermediates. In particular, recombinant Aβ studies described Aβ hexamers as critical oligomeric nuclei. We recently identified hexameric Aβ assemblies in a cellular model, and revealed their ability to enhance recombinant Aβ aggregation in vitro. Here, we assessed the contribution of similar hexameric-like Aβ assemblies to the development of amyloid pathology. We report their early presence in both transgenic mice brains exhibiting human Aβ pathology and cerebrospinal fluid of AD patients, suggesting hexameric Aβ as a putative novel AD biomarker. Using isolated cell-derived hexameric Aβ, we report the potential of these assemblies to seed other human Aβ species, resulting in neuronal toxicity in vitro and amyloid deposition aggravation in vivo. In order to identify key contributors to their formation in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in the formation of hexameric-like Aβ assemblies. As catalytic subunits of the γ-secretase complex, PS1 and PS2 can differentially participate in Aβ generation. Using CRISPR-Cas9-modified neuronal-like cell lines knockdown for each of the two presenilins, we present experimental evidence suggesting a direct link between the PS2-dependent pathway and the release of hexameric-like Aβ assemblies in extracellular vesicles.

Recombinant human BMP-4 growth factor (GF) has significant commercial potential as therapeutic for regenerating bone and as cell culture supplement. However, its commercial utility has been limited as large-scale attempts to express and purify human BMP-4 GF have proved challenging. We have established a novel approach to obtain significant quantities of pure and bioactive BMP-4 GF from Chinese hamster ovary cell cultures by extracting the GF moiety from the extracellular matrix or cell pellet fraction. This approach increased yields approximately one 100-fold over BMP-4 GF purified from CM. The molecular activities of the two fractions are indistinguishable. We further analyzed binding of BMP-4 GF to the proteoglycan Heparin and showed that an N-terminal basic sequence is essential for this interaction. Taken together, these results provide novel insights into the purification, localization, and Heparin binding of human BMP-4 that have implications for its bioprocessing and biological function.

In humans, cellular mechanoperception serves as the basis of touch sensation and proprioception, contributes to the proper programming of cell fate during embryonic development, and plays a pivotal role in the development of mechanosensitive tissues. Molecular mechanoreceptors can respond to their environment by mediating transient adjustments of ion homeostasis, which subsequently trigger calcium-dependent alteration of gene expression via specific signaling pathways such as the NFAT (nuclear factor of activated T-cells) pathway. Although, mechanoreceptors are potential drug targets for various diseases, current techniques to study mechanically gated processes are often based on custom tailored microfluidic systems, which require special setups or have limited throughput. Here, we present a platform to characterize shear-stress-triggered, calcium-mediated gene expression, which employs a programmable, 96-well-format, shear-stress induction device to examine the effects of imposing various mechanical loads on mammalian adherent cell lines. The presented method is suitable for high-throughput experiments and provides a large tunable parameter space to optimize conditions for different cell types. Our findings indicate that the device is an effective tool to explore conditions in terms of frequency, intensity, intervals as well as extracellular matrix composition alongside the evaluation of different combinations of mechanosensitive proteins for mechanically activated gene expression. We believe our results can serve as a platform for further investigations into shear stress-controlled gene expression in basic research and drug screening. This article is protected by copyright. All rights reserved.

Ozanimod, a sphingosine 1-phosphate (S1P) receptor modulator that binds with high affinity selectively to S1P receptor subtypes 1 (S1P1) and 5 (S1P5), is approved for the treatment of relapsing multiple sclerosis (MS) in multiple countries. Ozanimod profiling revealed a species difference in its potency for S1P5 in mouse, rat, and canine compared with that for human and monkey. Site-directed mutagenesis identified amino acid alanine at position 120 to be responsible for loss of activity for mouse, rat, and canine S1P5, and mutation back to threonine as in human/monkey S1P5 restored activity. Radioligand binding analysis performed with mouse S1P5 confirmed the potency loss is a consequence of a loss of affinity of ozanimod for mouse S1P5 and was restored with mutation of alanine 120 to threonine. Study of ozanimod in preclinical mouse models of MS can now determine the S1P receptor(s) responsible for observed efficacies with receptor engagement as measured using pharmacokinetic exposures of free drug. Hence, in the experimental autoimmune encephalomyelitis model, ozanimod exposures sufficient to engage S1P1, but not S1P5, resulted in reduced circulating lymphocytes, disease scores, and body weight loss; reduced inflammation, demyelination, and apoptotic cell counts in the spinal cord; and reduced circulating levels of the neuronal degeneration marker, neurofilament light. In the demyelinating cuprizone model, ozanimod prevented axonal degradation and myelin loss during toxin challenge but did not facilitate enhanced remyelination after intoxication. Since free drug levels in this model only engaged S1P1, we concluded that S1P1 activation is neuroprotective but does not appear to affect remyelination. SIGNIFICANCE STATEMENT Ozanimod, a selective modulator of human sphingisone 1-phosphate receptor subtypes 1 and 5 (S1P1/5), displays reduced potency for rodent and dog S1P5 compared with human, which results from mutation of threonine to alanine at position 120. Ozanimod can thus be used as a selective S1P1 agonist in mouse models of multiple sclerosis to define efficacies driven by S1P1 but not S1P5. Based on readouts for experimental autoimmune encephalomyelitis and cuprizone intoxication, S1P1 modulation is neuroprotective, but S1P5 activity may be required for remyelination.

A transferrin receptor (TfR)-targeted nanodrug [green fluorescence emission carbon dot (GCD)-polyethylene glycol (PEG)-transferrin (Tf)@doxorubicin (Dox)] for cancer therapy was developed by functionalizing GCDs with PEG, Tf, and Dox. GCDs were synthesized by the one-step hydrothermal method, followed by conjugating PEG and Tf by covalent bonds and loading Dox by electrostatic interactions. The nanodrug exhibits high stability under neutral conditions and effectively releases Dox at pH of 5.5. GCD-PEG-Tf@Dox can be selectively internalized by TfR-overexpressed tumor cells (MCF-7 and K150) via receptor-mediated endocytosis and further release Dox to the nuclei. As a result, GCD-PEG-Tf@Dox exhibits significant lethality to tumor cells (MCF-7 and K150) but greatly reduced toxicity to normal cells [Chinese hamster ovary cell line (CHO)] compared with free Dox. In vivo studies have confirmed that GCD-PEG-Tf@Dox can effectively inhibit tumor proliferation with negligible side effects.

Background: Pulmonary surfactant dysfunction is an important pathological factor in acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF). Objective: In this study, the characteristics of recombinant mature surfactant protein B (SP-B) and reteplase (rPA) fusion protein maintaining good pulmonary surface activity and rPA fibrinolytic activity in acute lung injury cell model were studied. Methods: We studied the characteristics of SP-B fusion expression, cloned rPA gene and N-terminal rPA/C-terminal SP-B co-expression gene, and constructed them into eukaryotic expression vector pEZ-M03 to obtain recombinant plasmids pEZ-rPA and pEZ-rPA/SP-B. The recombinant plasmids was transfected into Chinese hamster ovary (CHO) K1 cells and the expression products were analyzed by Western Blot. Lipopolysaccharide (LPS) was used to induce CCL149 (an alveolar epithelial cell line) cell injury model. Fluorescence staining of rPA and rPA/SP-B was carried out with the enhanced green fluorescent protein (eGFP) that comes with pEZ-M03; the cell Raman spectroscopy technique was used to analyze the interaction between rPA/SP-B fusion protein and the phospholipid structure of cell membrane in CCL149 cells. The enzyme activity of rPA in the fusion protein was determined by fibrin-agarose plate method. Results: The rPA/SP-B fusion protein was successfully expressed. In the CCL149 cell model of acute lung injury (ALI), the green fluorescence of rPA/SP-B is mainly distributed on the CCL149 cell membrane. The rPA/SP-B fusion protein can reduce the disorder of phospholipid molecules and reduce cell membrane damage. The enzyme activity of rPA/SP-B fusion protein was 3.42, and the fusion protein still had good enzyme activity. Conclusion: The recombinant eukaryotic plasmid pEZ-rPA/SP-B is constructed and can be expressed in the eukaryotic system. Studies have shown that rPA/SP-B fusion protein maintains good SP-B lung surface activity and rPA enzyme activity in acute lung injury cell model.

The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies have shown high promise for this protein as a vaccine against HIV. However, one of the main challenges with manufacturing this promising protein has been ascribed to the low yield obtained in mammalian cell cultures. Significant improvements in gp145 production are needed to address this issue to test the gp145 protein as a potentially effective, safe, and affordable HIV vaccine. Here we describe the application of a novel expression technology to create GMP-grade CHO cell lines expressing approximately 50 μg/ml in non-optimized fed-batch culture, which is an order of magnitude higher than that obtained in existing processes. Top producing clones show a high degree of similarity in the glycosylation patterns of the purified protein to the reference standard. Conformational integrity and functionality was demonstrated via high-affinity binding to soluble CD4, using a panel of antibodies including VRC01, F105, Hk20, PG9 and 17b. In summary, we were able to generate CHO cell lines expressing HIV gp145 with significantly higher overall expression yields than currently accessible, and high product quality that could potentially be suitable for future studies assessing the efficacy and safety of gp145-based HIV vaccines.

3-Hydroxy-3-methyl glutaryl-coenzyme A reductase (Hmgcr) encodes the rate-limiting enzyme in the cholesterol biosynthesis pathway. The regulation of Hmgcr in rat models of genetic hypertension (viz. Spontaneously Hypertensive Rat [SHR] and its normotensive control Wistar/Kyoto [WKY] strain) is unclear. Interestingly, Hmgcr transcript and protein levels are diminished in liver tissues of SHR as compared to WKY. This observation is consistent with the diminished plasma cholesterol level in SHR animals. However, the molecular basis of these apparently counter-intuitive findings remains completely unknown. Sequencing of the Hmgcr promoter in SHR and WKY strains reveals three variations: A-405G, C-62T and a 11 bp insertion (‐398_-388insTGCGGTCCTCC) in SHR. Among these variations, A-405G occurs at an evolutionarily-conserved site among many mammals. Moreover, SHR-Hmgcr promoter displays lower activity than WKY-Hmgcr promoter in various cell lines. Transient transfections of Hmgcr-promoter mutants and in silico analysis suggest altered binding of Runx3 and Srebf1 across A-405G site. On the other hand, C-62T and -398_-388insTGCGGTCCTCC variations do not appear to contribute to the reduced Hmgcr promoter activity in SHR as compared to WKY. Indeed, chromatin immunoprecipitation assays confirm differential binding of Runx3 and Srebf1 to Hmgcr promoter leading to reduced expression of Hmgcr in SHR as compared to WKY under basal as well as cholesterol-modulated conditions. Taken together, this study provides, for the first time, molecular basis for diminished Hmgcr expression in SHR animals, which may account for the reduced circulating cholesterol level in this widely-studied model for cardiovascular diseases.

Cellular DNA is composed of four canonical nucleosides (dA, dC, dG and T), which form two Watson–Crick base pairs. In addition, 5‐methylcytosine (mdC) may be present. The methylation of dC to mdC is known to regulate transcriptional activity. Next to these five nucleosides, the genome, particularly of stem cells, contains three additional dC derivatives, which are formed by stepwise oxidation of the methyl group of mdC with the help of Tet enzymes. These are 5‐hydroxymethyl‐dC (hmdC), 5‐formyl‐dC (fdC), and 5‐carboxy‐dC (cadC). It is believed that fdC and cadC are converted back into dC, which establishes an epigenetic control cycle that starts with methylation of dC to mdC, followed by oxidation and removal of fdC and cadC. While fdC was shown to undergo intragenomic deformylation to give dC directly, a similar decarboxylation of cadC was postulated but not yet observed on the genomic level. By using metabolic labelling, we show here that cadC decarboxylates in several cell types, which confirms that both fdC and cadC are nucleosides that are directly converted back to dC within the genome by C−C bond cleavage., A possibility that 5‐carboxydC (cadC), which is a modification found in somatic and stem cells, is directly decarboxylated to dC via C−C bond cleavage in cells is suggested.

Purpose A major difficulty in monoclonal antibody (mAb) therapeutic development is product aggregation. In this study, intermolecular isopeptide bonds in mAb aggregates were characterized for the first time. We aim to propose a mechanism of covalent aggregation in a model antibody using stressed studies at raised temperatures to aid in the understanding of mAb aggregation pathways. Methods Aggregate fractions were generated using raised temperature and were purified using size-exclusion chromatography (SEC). The fractions were tryptically digested and characterized using liquid chromatography hyphenated to tandem mass-spectrometry (LC–MS/MS). Results An increased amount of clipping between aspartic acid and proline in a solvent accessible loop in the constant heavy 2 (CH2) domain of the mAb was observed under these conditions. Detailed peptide mapping revealed 14 isopeptide bonds between aspartic acid at that cleavage site and lysine residues on adjacent antibodies. Two additional isopeptide bonds were identified between the mAb HC N-terminal glutamic acid or a separate aspartic acid to lysine residues on adjacent antibodies. Conclusions Inter-protein isopeptide bonds between the side chains of acidic amino acids (aspartate and glutamate) and lysine were characterized for the first time in mAb aggregates. A chemical mechanism was presented whereby spontaneous isopeptide bond formation could be facilitated via either the aspartic acid side chain or C-terminus.

The majority of depleting monoclonal antibody (mAb) drugs elicit responses via Fc-FcγR and Fc-C1q interactions. Optimal C1q interaction is achieved through hexameric Fc:Fc interactions at the target cell surface. Herein is described an approach to exploit the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Fusion of the C-terminal tailpiece of IgM promoted spontaneous hIgG hexamer formation, resulting in enhanced C1q recruitment and complement-dependent cytotoxicity (CDC) but with off-target complement activation and reduced in-vivo efficacy. Mutation of the penultimate tailpiece cysteine to serine (C575S) ablated spontaneous hexamer formation, but facilitated reversible hexamer formation after concentration in solution. C575S mutant tailpiece antibodies displayed increased complement activity only after target binding, in-line with the concept of ‘on-target hexamerisation’, whilst retaining efficient in-vivo efficacy and augmented target cell killing in the lymph node. Hence, C575S-tailpiece technology represents an alternative format for promoting on-target hexamerisation and enhanced CDC., Sopp et al describe an approach, which exploits the tailpiece of the naturally multimeric IgM to augment hexamerisation of IgG. Their approach provides a newly engineered format of antibodies for promoting hexamerisation and enhanced complement-dependent cytotoxicity only when cell surface bound.

Chinese hamster ovary (CHO) cells present inherent limitations for processing and secretion of large amounts of recombinant proteins, especially for those requiring complex post-translational processing. To tackle these limitations, we engineered CHO host cells (CHOK1 and CHOS) to overexpress the transcription factor BLIMP1/prdm1 (a master regulator of the highly-secreting phenotype of antibody-producing plasma cells), generating novel CHO cell lines (referred to as CHOB). The CHOB cell lines exhibited decreased cell densities, prolonged stationary phase and arrested cell cycle in G1/G0 phase but simultaneously had significantly greater product titre for recombinant IgG1 (> 2-fold increase) coupled with a significantly greater cell-specific productivities (> 3-fold increase). We demonstrated that the improved productive phenotype of CHOB cells resulted from a series of changes to cell physiology and metabolism. CHOB cells showed a significantly greater ER size and increased protein synthesis and secretion capacity compared to control cells. In addition, CHOB cells presented a metabolic profile that favoured energy production to support increased recombinant protein production. This study indicated that a cell engineering approach based on BLIMP1 expression offers great potential for improving the secretory capacity of CHO cell hosts utilised for manufacture of recombinant biopharmaceuticals. Our findings also provides a greater understanding of the relationship between cell growth and productivity, valuable generic information for improving productive phenotypes for CHO cell lines during industrial cell line development.

Ethylene (ET) is an important gaseous plant hormone. It is highly desirable to develop fluorescent probes for monitoring ethylene in living cells. We report an efficient RhIII -catalysed coupling of N-phenoxyacetamides to ethylene in the presence of an alcohol. The newly discovered coupling reaction exhibited a wide scope of N-phenoxyacetamides and excellent regioselectivity. We successfully developed three fluorophore-tagged RhIII -based fluorogenic coumarin-ethylene probes (CEPs) using this strategy for the selective and quantitative detection of ethylene. CEP-1 exhibited the highest sensitivity with a limit of detection of ethylene at 52 ppb in air. Furthermore, CEP-1 was successfully applied for imaging in living CHO-K1 cells and for monitoring endogenous-induced changes in ethylene biosynthesis in tobacco and Arabidopsis thaliana plants. These results indicate that CEP-1 has great potential to illuminate the spatiotemporal regulation of ethylene biosynthesis and ethylene signal transduction in living biological systems.

The cell cycle is a sequential multi-step process essential for growth and proliferation of cells comprising multicellular organisms. Although a number of proteins are known to modulate the cell cycle, the role of lipids in regulation of cell cycle is still emerging. In our previous work, we monitored the role of cholesterol in cell cycle progression in CHO-K1 cells. Since sphingolipids enjoy a functionally synergistic relationship with membrane cholesterol, in this work, we explored whether sphingolipids could modulate the eukaryotic cell cycle using CHO-K1 cells. Sphingolipids are essential components of eukaryotic cell membranes and are involved in a number of important cellular functions. To comprehensively monitor the role of sphingolipids on cell cycle progression, we carried out metabolic depletion of sphingolipids in CHO-K1 cells using inhibitors (fumonisin B1, myriocin, and PDMP) that block specific steps of the sphingolipid biosynthetic pathway and examined their effect on individual cell cycle phases. Our results show that metabolic inhibitors led to significant reduction in specific sphingolipids, yet such inhibition in sphingolipid biosynthesis did not show any effect on cell cycle progression in CHO-K1 cells. We speculate that any role of sphingolipids on cell cycle progression could be context and cell-type dependent, and cancer cells could be a better choice for monitoring such regulation, since sphingolipids are differentially modulated in these cells.

ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for anti-proliferative effects. Curve-shift experiments using D2R-mediated b-arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC2019s functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. Significance Statement ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. In this study, we demonstrate that it antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anti-cancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders.

Chinese hamster ovary (CHO) cells are regarded as a prominent host for manufacturing therapeutic proteins. Although conventional strategies for generating recombinant proteins in CHO cells depend on the random integration of a gene of interest (GOI), these established techniques occasionally result in genetically heterogeneous cell lines, which causes diminished expression of the recombinant proteins in the long run. Production instability can be reduced by SSI and creates stable cell lines with a consistent expression of the GOI. In this experiment, we demonstrate the targeted incorporation of a reporter cassette in two PhiC31 pseudo attP sites of CHO cells exploiting the homology-directed repair (HDR) generated by the CRISPR/Cas9 platform. Genes encoding GFP and puromycin resistance marker were precisely inserted into these loci via CRISPR/Cas9. Stable cell lines were suitably produced following antibiotic selection. Junction PCR and fluorescence assay determined targeted integration and expression homogeneity of the reporter cassette, respectively. Taken together, our results indicate the possibility of these two PhiC31 pseudo attP sites as the target sites for site-specific integration of a transgene mediated by CRISPR/Cas9. Furthermore, higher knock-in efficiency and expression homogeneity was observed in the pseudo attP site associated with chromosome 6 compared to the pseudo attP site from chromosome 3.

Nanosecond electric pulses have several potential advantages in electroporation-based procedures over the conventional micro- and millisecond pulses including low level of heating, reduced electrochemical reactions and reduced muscle contractions making them alluring for use in biomedicine and food industry. The aim of this study was to evaluate if nanosecond pulses can enhance the cytotoxicity of chemotherapeutics bleomycin and cisplatin in vitro and to quantify metal release from electrodes in comparison to 100 μs pulses commonly used in electrochemotherapy. The effects of nanosecond pulse parameters (voltage, pulse duration, number of pulses) on cell membrane permeabilization, resealing and on cell survival after electroporation only and after electrochemotherapy with bleomycin and cisplatin were evaluated on Chinese hamster ovary cells. Application of permeabilizing nanosecond pulses in combination with chemotherapeutics resulted in successful cell kill. Higher extracellular concentrations of bleomycin – but not cisplatin – were needed to achieve the same decrease in cell survival with nanosecond pulses as with eight 100 μs pulses, however, the tested bleomycin concentrations were still considerably lower compared to doses used in clinical practice. Decreasing the pulse duration from microseconds to nanoseconds and concomitantly increasing the amplitude to achieve the same biological effect resulted in reduced release of aluminum ions from electroporation cuvettes.

Mucin-type O-glycosylation (O-glycosylation) is a common post-translational modification that confers distinct biophysical properties to proteins and plays crucial roles in intercellular signaling. Yet, despite the importance of O-glycans, relatively few tools exist for their analysis and modification. In particular, there is a need for enzymes that can cleave the wide range of O-glycan structures found on protein surfaces, to facilitate glycan profiling and editing. Through functional metagenomic screening of the human gut microbiome, we discovered endo-O-glycan hydrolases from CAZy family GH101 that are capable of slowly cleaving the intact sialyl T-antigen trisaccharide (a ubiquitous O-glycan structure in humans) in addition to their primary activity against the T-antigen disaccharide. We then further explored this sequence space through phylogenetic profiling and analysis of representative enzymes, revealing large differences in the levels of this promiscuous activity between enzymes within the family. Through structural and sequence analysis, we identified active site residues that modulate specificity. Through subsequent rational protein engineering, we improved the activity of an enzyme identified by phylogenetic profiling sufficiently that substantial removal of the intact sialyl T-antigen from proteins could be readily achieved. Our best sialyl T-antigen hydrolase mutant, SpGH101 Q868G, is further shown to function on a number of proteins, tissues, and cells. Access to this enzyme opens up improved methodologies for unraveling the glycan code.

Tumor necrosis factor-alpha (TNF⍺) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNF⍺ inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNF⍺ receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNF⍺ more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNF⍺ (Kd 279±40.9nM) and MTV-LRP1 (Kd 399±50.5nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNF⍺ with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders. This article is protected by copyright. All rights reserved.

In the wake of the COVID-19 pandemic, drug repurposing has been highlighted for rapid introduction of therapeutics. Proposed drugs with activity against SARS-CoV-2 include compounds with positive charges at physiologic pH, making them potential targets for the organic cation secretory transporters of kidney and liver, i.e., the basolateral organic cation transporters, OCT1 and OCT2; and the apical multidrug and toxin extruders, MATE1 and MATE2-K. We selected several compounds proposed to have in vitro activity against SARS-CoV-2 (chloroquine, hydroxychloroquine, quinacrine, tilorone, pyronaridine, cetylpyridinium, and miramistin) to test their interaction with OCT and MATE transporters. We used Bayesian machine learning models to generate predictions for each molecule with each transporter and also experimentally determined IC(50) values for each compound against labeled substrate transport into CHO cells that stably expressed OCT2, MATE1, or MATE2-K using three structurally distinct substrates (atenolol, metformin and 1-methyl-4-phenylpyridinium) to assess the impact of substrate structure on inhibitory efficacy. For the OCTs substrate identity influenced IC(50) values, although the effect was larger and more systematic for OCT2. In contrast, inhibition of MATE1-mediated transport was largely insensitive to substrate identity. Unlike MATE1, inhibition of MATE2-K was influenced, albeit modestly, by substrate identity. Maximum unbound plasma concentration/IC(50) ratios were used to identify potential clinical DDI recommendations; all the compounds interacted with the OCT/MATE secretory pathway, most with sufficient avidity to represent potential DDI issues for secretion of cationic drugs. This should be considered when proposing cationic agents as repurposed antivirals. SIGNIFICANCE STATEMENT: Drugs proposed as potential COVID-19 therapeutics based on in vitro activity data against SARS-CoV-2 include compounds with positive charges at physiological pH, making them potential interactors with the OCT/MATE renal secretory pathway. We tested seven such molecules as inhibitors of OCT1/2 and MATE1/2-K. All the compounds blocked transport activity regardless of substrate used to monitor activity. Suggesting that plasma concentrations achieved by normal clinical application of the test agents could be expected to influence the pharmacokinetics of selected cationic drugs.

Freezing of cell culture supernatant (CCS) is a standard procedure in process development of monoclonal antibody (mAb) platform processes as up- and downstream development are usually separated. In the manufacturing process of mAb, however, freezing is avoided, which poses the question of comparability and transferability from process development to manufacturing. In this case study, mAb CCS from Chinese hamster ovary (CHO) cells is frozen and thawed in a novel active freezing device and subsequently captured by protein A chromatography. Critical quality attributes such as host cell protein (HCP) concentration and soluble mAb dimer shares have been monitored throughout the case study. Furthermore, cryo-concentration of individual proteins was investigated. The main factors that drive cryo-concentration are diffusion and natural convection. Natural convection in freezing processes was found to increase at warmer freezing temperatures and thus slower freezing, leading to higher concentration gradients from top to bottom of a freezing chamber. The freeze concentration was dependent on protein size and correlated to diffusivity, where smaller proteins are exposed to higher cryo-concentration. Our results suggest that as a result of freezing processes, large particles based on mAb and specific host cell proteins (HCPs) expressing a certain affinity to mAbs are formed that have to be removed before purification. This leads to a significant improvement in HCP reduction by the protein A step, when compared with reference samples, where twice as much HCP remained in the eluate. Furthermore, HCP and mAb dimer concentrations in protein A eluate were dependent on the freezing temperature. As a conclusion, CCS should be frozen as rapidly as possible during process development to minimize issues of transferability from process development to manufacturing.

We describe a systematic approach to establish predictive models of CHO cell growth, cell metabolism and monoclonal antibody (mAb) formation during biopharmaceutical production. The prediction is based on a combination of an empirical metabolic model connecting extracellular metabolic fluxes with cellular growth and product formation with mixed Monod-inhibition type kinetics that we generalized to every possible external metabolite. We describe the maximum specific growth rate as a function of the integral viable cell density (IVCD). Moreover, we also take into account the accumulation of metabolites in intracellular pools that can influence cell growth. This is possible even without identification and quantification of these metabolites as illustrated with fed-batch cultures of Chinese Hamster Ovary (CHO) cells producing a mAb. The impact of cysteine and tryptophan on cell growth and cell productivity was assessed, and the resulting macroscopic model was successfully used to predict the impact of new, untested feeding strategies on cell growth and mAb production. This model combining piecewise linear relationships between metabolic rates, growth rate and production rate together with Monod-inhibition type models for cell growth did well in predicting cell culture performance in fed-batch cultures even outside the range of experimental data used for establishing the model. It could therefore also successfully be applied for in silico prediction of optimal operating conditions.

A straightforward screening of a compound library comprising 2439 substances for the identification of new inhibitors for the neurotransmitter transporters GlyT1 and GlyT2 is described. Screening and full‐scale competition experiments were performed using recently developed GlyT1 and GlyT2 MS Binding Assays. That way for both targets, GlyT1 and GlyT2, ligands were identified, which exhibited affinities (pK i values) in the low micromolar to sub‐micromolar range. The majority of these binders exhibit new chemical scaffolds in the class of GlyT1 and GlyT2 inhibitors, which could be of interest for the development of new ligands with improved affinities for the target proteins. Additionally, compounds with excellent fluorescent properties were found for GlyT2, which renders them promising compounds for future fluorescence‐based techniques. All in all, this study demonstrates that MS Binding Assays represent a powerful technology platform also well suited for the screening of compound libraries in a highly reliable and effective manner., Reliable and effective: GlyT1 and GlyT2 MS Binding Assays were used for a library screening investigating 2439 compounds. For GlyT1 and GlyT2 several inhibitors were identified, of which GlyT2 Inhibitors exhibiting excellent fluorescent properties were of great interest. The outcome of this study demonstrated that MS Binding Assays are powerful tools for the examination of compound libraries of reasonable size in a straightforward manner.

Host cell proteins (HCP) that co-purify with biologics produced in Chinese hamster ovary cells have been shown to impact product quality through proteolytic degradation of recombinant proteins, leading to potential product losses. Several problematic HCPs can remain in the final product even after extensive purification. Each recombinant cell line has a unique HCP profile that can be determined by numerous upstream and downstream factors, including clonal variation and the protein sequence of the expressed therapeutic molecule. Here, we worked with recombinant cell lines with high levels of copurifying HCPs, and showed that in those cell lines even modest downregulation (≤50%) of the difficult to remove HCP Cathepsin D, through stable shRNA interference or monoallelic deletion of the target gene using CRISPR-Cas 9, is sufficient to greatly reduce levels of co-purifying HCP as measured by high throughput targeted LC-MS. This reduction led to improved product quality by reducing fragmentation of the drug product in forced degradation studies to negligible levels. We also show the potential of cell engineering to target other undesired HCPs and relieve the burden on downstream purification. This article is protected by copyright. All rights reserved.

Commercial production of therapeutic proteins using mammalian cells requires complex process solutions, and consistency of these process solutions is critical to maintaining product titer and quality between batches. Inconsistencies between process solutions prepared at bench and commercial scale may be due to differences in mixing time, temperature, and pH which can lead to precipitation and subsequent removal via filtration of critical solution components such as trace metals. Pourbaix diagrams provide a useful tool to model the solubility of trace metals and were applied to troubleshoot the scale-up of nutrient feed preparation after inconsistencies in product titer were observed between bench- and manufacturing-scale batches. Pourbaix diagrams modeled the solubility of key metals in solution at various stages of the nutrient feed preparation and identified copper precipitation as the likely root cause of inconsistent medium stability at commercial scale. Copper precipitation increased proportionally with temperature in bench-scale preparations of nutrient feed and temperature was identified as the root cause of copper precipitation at the commercial scale. Additionally, cell culture copper titration studies performed in bench-scale bioreactors linked copper-deficient mammalian cell culture to inconsistent titers at the commercial scale. Pourbaix diagrams can predict when trace metals are at risk of precipitating and can be used to mitigate risk during the scale-up of complex medium preparations.

Loss of orexin-producing neurons results in narcolepsy with cataplexy, and orexin agonists have been shown to increase wakefulness and alleviate narcolepsy symptoms in animal models. Several OX2R agonists have been reported but with little or no activity at OX1R. We conducted structure-activity relationship studies on the OX2R agonist YNT-185 (2) and discovered dual agonists such as RTOXA-43 (40) with EC50's of 24 nM at both OX2R and OX1R. Computational modeling studies based on the agonist-bound OX2R cryogenic electron microscopy structures showed that 40 bound in the same binding pocket and interactions of the pyridylmethyl group of 40 with OX1R may have contributed to its high OX1R potency. Intraperitoneal injection of 40 increased time awake, decreased time asleep, and increased sleep/wake consolidation in 12-month old mice. This work provides a promising dual small molecule agonist and supports development of orexin agonists as potential treatments for orexin-deficient disorders such as narcolepsy.

Exocyst complex component 3-like 1 (EXOC3L1), which regulates insulin secretion, is ubiquitously present in heart, lung, liver, spleen, kidney, muscle, cerebellum, pituitary, adrenal grand, and pancreatic islets. Its deduced amino acid sequence has 31% identity and 53% similarity with Sec6, so they are considered isoforms. Since Sec6 suppresses apoptosis via HSP27, we investigated the involvement of EXOC3L1 expression in apoptosis. We found that overexpressed EXOC3L1 in Chinese hamster ovary cells significantly reduced cultured cell numbers. It also significantly increased apoptotic DNA ladder, caspase 3 activity, and cleavage of caspase 3 compared with the control. Thus, although Sec6 reduces apoptosis by increasing HSP27 phosphorylation, overexpressed EXOC3L1 alone can spontaneously induce apoptosis without apoptotic stimulators or inducers.

Therapeutic antibodies are decorated with complex-type N-glycans that significantly affect their biodistribution and bioactivity. The N-glycan structures on antibodies are incompletely processed in wild-type CHO cells due to their limited glycosylation capacity. To improve N-glycan processing, glycosyltransferase genes have been traditionally overexpressed in CHO cells to engineer the cellular N-glycosylation pathway by using random integration, which is often associated with large clonal variations in gene expression levels. In order to minimize the clonal variations, we used recombinase-mediated-cassette-exchange (RMCE) technology to overexpress a panel of 42 human glycosyltransferase genes to screen their impact on antibody N-linked glycosylation. The bottlenecks in the N-glycosylation pathway were identified and then released by overexpressing single or multiple critical genes. Overexpressing B4GalT1 gene alone in the CHO cells produced antibodies with more than 80% galactosylated bi-antennary N-glycans. Combinatorial overexpression of B4GalT1 and ST6Gal1 produced antibodies containing more than 70% sialylated bi-antennary N-glycans. In addition, antibodies with various tri-antennary N-glycans were obtained for the first time by overexpressing MGAT5 alone or in combination with B4GalT1 and ST6Gal1. The various N-glycan structures and the method for producing them in this work provide opportunities to study the glycan structure-and-function and develop novel recombinant antibodies for addressing different therapeutic applications.

Accumulation of amyloid-β peptide (Aβ) in neuronal cells and in the extracellular regions in the brain is a major cause of Alzheimer's disease (AD); therefore, inhibition of Aβ accumulation offers a promising approach for therapeutic strategies against AD. Aβ is produced by sequential proteolysis of amyloid precursor protein (APP) in late/recycling endosomes after endocytosis of APP located in the plasma membrane. Aβ is then released from cells in a free form or in an exosome-bound form. Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli. Recently, we found that one of the Stx subtypes, Stx2a, has a unique intracellular transport route after endocytosis through its receptor-binding B-subunit. A part of Stx2a can be transported to late/recycling endosomes and then degraded in a lysosomal acidic compartment, although in general Stx is transported to the Golgi and then to the endoplasmic reticulum in a retrograde manner. In this study, we found that treatment of APP-expressing cells with a mutant Stx2a (mStx2a), lacking cytotoxic activity because of mutations in the catalytic A-subunit, stimulated the transport of APP to the acidic compartment, which led to degradation of APP and a reduction in the amount of Aβ. mStx2a-treatment also inhibited the extracellular release of Aβ. Therefore, mStx2a may provide a new strategy to inhibit the production of Aβ by modulating the intracellular transport of APP.

The deposition of tau aggregates throughout the brain is a pathological characteristic within a group of neurodegenerative diseases collectively termed tauopathies, which includes Alzheimer’s disease. While recent findings suggest the involvement of unconventional secretory pathways driving tau into the extracellular space and mediating the propagation of the disease-associated pathology, many of the mechanistic details governing this process remain elusive. In the current study, we provide an in-depth characterization of the unconventional secretory pathway of tau and identify novel molecular determinants that are required for this process. Here, using Drosophila models of tauopathy, we correlate the hyperphosphorylation and aggregation state of tau with the disease-related neurotoxicity. These newly established systems recapitulate all the previously identified hallmarks of tau secretion, including the contribution of tau hyperphosphorylation as well as the requirement for PI(4,5)P2 triggering the direct translocation of tau. Using a series of cellular assays, we demonstrate that both the sulfated proteoglycans on the cell surface and the correct orientation of the protein at the inner plasma membrane leaflet are critical determinants of this process. Finally, we identify two cysteine residues within the microtubule binding repeat domain as novel cis-elements that are important for both unconventional secretion and trans-cellular propagation of tau.

The glucagon-like peptide-1 (GLP-1) receptor is a validated drug target for metabolic disorders. Ago-allosteric modulators are capable of acting both as agonists on their own and as efficacy enhancers of orthosteric ligands. However, the molecular details of ago-allosterism remain elusive. Here, we report three cryo-electron microscopy structures of GLP-1R bound to (i) compound 2 (an ago-allosteric modulator); (ii) compound 2 and GLP-1; and (iii) compound 2 and LY3502970 (a small molecule agonist), all in complex with heterotrimeric Gs. The structures reveal that compound 2 is covalently bonded to C347 at the cytoplasmic end of TM6 and triggers its outward movement in cooperation with the ECD whose N terminus penetrates into the GLP-1 binding site. This allows compound 2 to execute positive allosteric modulation through enhancement of both agonist binding and G protein coupling. Our findings offer insights into the structural basis of ago-allosterism at GLP-1R and may aid the design of better therapeutics., The glucagon-like peptide-1 (GLP-1) receptor is a key regulator of glucose homeostasis and a drug target for type 2 diabetes but available GLP-1R agonists are suboptimal due to several side-effects. Here authors report the cryo-EM structure of GLP-1R bound to an ago-allosteric modulator in complex with heterotrimeric Gs which offers insights into the molecular details of ago-allosterism.

WNT5A activates noncanonical Wnt signaling pathways and has critical functions in early development, differentiation, and tissue homeostasis. Two major WNT5A protein isoforms, which in this study we term WNT5A‐L(A) and WNT5A‐S(B), have been identified that differ by 18 AA at their amino terminus. Functional differences between the isoforms have been indicated in studies utilizing cancer cell lines but the activities of the isoforms in normal cells and during differentiation have not been explored. We examined the WNT5A isoforms in the normal osteoblast cell line hFOB1.19. WNT5A‐L(A) and WNT5A‐S(B) transcripts increased from Days 3 to 21 of differentiation but WNT5A‐S(B) showed a greater fold‐change. In undifferentiated cells, there are 2‐fold more WNT5A‐L(A) than WNT5A‐S(B) transcripts. Total intracellular WNT5A protein increased up to 3‐fold during differentiation. siRNA knockdown of total WNT5A leads to a decrease in the expression of the differentiation markers, osteocalcin and RUNX2. Conditioned medium containing the isoform proteins [CM‐L(A) and CM‐S(B)] was used to analyze the effects of the isoforms on β‐catenin and noncanonical signaling, proliferation, gene expression, and alkaline phosphatase (ALP) activity. Treatment with both CM‐L(A) and CM‐S(B) reduced β‐catenin signaling. CM‐L(A) but not CM‐S(B) significantly increased the proliferation of nondifferentiated hFOB1.19 cells. CM‐L(A) enhanced osteocalcin transcripts over 2‐fold in differentiating cells, whereas CM‐S(B) had no effect. Analysis of differentiating cells up to Day 21 revealed no significant effect of treatment with CM‐L(A) or CM‐S(B) on ALP activity or osteocalcin gene expression. pJNK levels were unaffected in proliferating cells by treatment with neither isoform. pPKC increased slightly in CM‐L(A)‐treated cells at 15 min but by 2 h pPKC levels were less than the control. CM‐S(B) had a more robust effect on pPKC levels that continued up to 2 h. Together these results suggest that the WNT5A isoforms have distinct and overlapping functions in normal osteoblasts., The WNT5A ligand activates noncanonical Wnt signaling pathways important in development, differentiation, and cellular homeostasis. The activities of the two major isoforms of WNT5A were analyzed in normal osteoblasts. The isoforms were found to differentially affect proliferation, osteocalcin gene expression, and protein kinase C (PKC) activation but both reduce beta‐catenin signaling.

Residence time distribution modeling of integrated perfusion to capture process can elucidate the impact of product quality excursions and filter fouling on monoclonal antibody production. In this case study, a glycosylation inhibitor and fluorescently labeled antibody are applied to the continuous process to study protein quality modulation, perfusion filter fouling, and unit operation hold times. The unit operations were modeled as continuous-stirred tank reactors and the residence time distribution of a small molecule glycan inhibitor and impact on glycosylation were characterized. A fluorescently labeled antibody was applied as a tracer molecule and confirmed the impact of packed cell volume and filter fouling. This study demonstrates how a biologics continuous process can be modeled and characterized through residence time distribution to ensure a robust, well-understood process.