Search

This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG

Glycans as sugar polymers are important metabolic, structural, and physiological regulators for cellular and biological functions. They are often classified as critical quality attributes to antibodies and recombinant fusion proteins, given their impacts on the efficacy and safety of biologics drugs. Recent reports on the conjugates of N-acetyl-galactosamine and mannose-6-phosphate for lysosomal degradation, Fab glycans for antibody diversification, as well as sialylation therapeutic modulations and O-linked applications, have been fueling the continued interest in glycoengineering. The current advancements of the human glycome and the development of a comprehensive network in glycosylation pathways have presented new opportunities in designing next-generation therapeutic proteins.

Large-scale transient expression in Chinese Hamster Ovary (CHO) cells provides a rapid protein production method with a potential start-to-end alignment advantage for biotherapeutics drug discovery. In this chapter, experimental protocols are illustrated for transient expression of therapeutic glycoproteins with improved galactosylation and sialylation in ExpiCHO-S™ system. To reduce the production cost, we also describe a novel procedure for PEI-mediated transfection in ExpiCHO-S™ cells that supports therapeutic protein expression comparable to the level with ExpiFectamine™-based transfection.

Large-scale transient expression in Chinese Hamster Ovary (CHO) cells provides a rapid protein production method with a potential start-to-end alignment advantage for biotherapeutics drug discovery. In this chapter, experimental protocols are illustrated for transient expression of therapeutic glycoproteins with improved galactosylation and sialylation in ExpiCHO-S™ system. To reduce the production cost, we also describe a novel procedure for PEI-mediated transfection in ExpiCHO-S™ cells that supports therapeutic protein expression comparable to the level with ExpiFectamine™-based transfection.

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and β-glucuronidase assays in vitro and distinct properties in vivo. Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N′-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder in humans characterized by complex behavioral deficits, including intellectual disability, impaired social interactions and hyperactivity. ASD exhibits a strong genetic component with underlying multi-gene interactions. Candidate gene studies have shown that the neurobeachin gene is disrupted in human patients with idiopathic autism (Castermans et al., 2003). The gene for neurobeachin (NBEA) spans the common fragile site FRA 13A and encodes a signal scaffold protein (Savelyeva et al., 2006). In mice, NBEA has been shown to be involved in the trafficking and function of a specific subset of synaptic vesicles. (Medrihan et al., 2009; Savelyeva, Sagulenko, Schmitt, & Schwab, 2006). rugose (rg) is the Drosophila homologue of the mammalian and human neurobeachin. Our previous genetic and molecular analyses have shown that rg encodes an A kinase anchor protein (DAKAP 550), which interacts with components of the EGFR and Notch mediated signaling pathways, facilitating cross-talk between these and other pathways (Shamloula et al., 2002). We now present functional data from studies on the larval neuromuscular junction that reveal abnormal synaptic architecture and physiology. In addition, adult rg loss-of-function mutants exhibit defective social interactions, impaired habituation, aberrant locomotion and hyperactivity. These results demonstrate that Drosophila neurobeachin (rugose) mutants exhibit phenotypic characteristics reminiscent of human ASD and thus could serve as a genetic model for studying autism spectrum disorders.

Advancement in high-throughput screening methods of novel therapeutic proteins for early stage research and development, specifically mAbs, have given mid-scale (milligram to gram scale) purification groups access to more of these molecules. The available purification technologies built to support mid-scale production was not efficient or versatile enough to keep up with this surge. To remedy this problem, we have designed and built a custom instrument using an AKTA Pure. This system enables parallel processing up to 5 samples and has the versatility to perform 1- to 3-step purification processes in a single queue. Furthermore, a unique purification scheme can be selected for each of the five samples in the queue. Overall processing time has reduced by 83% compared to manual, non-parallel load methods. Here, we describe our novel approach and demonstrate the flexibility, speed and efficiency of the instrument.

As the antibody-drug conjugate (ADC) field grows increasingly important for cancer treatment, it is vital for researchers to establish a firm understanding of how ADCs function at the molecular level. To gain insight into ADC uptake, trafficking, and catabolism-processes that are critical to ADC efficacy and toxicity-imaging studies have been performed with fluorophore-labeled conjugates. However, such labels may alter the properties and behavior of the ADC under investigation. As an alternative approach, we present here the development of a "clickable" ADC bearing an azide-functionalized linker-payload (LP) poised for "click" reaction with alkyne fluorophores; the azide group represents a significantly smaller structural perturbation to the LP than most fluorophores. Notably, the clickable ADC shows excellent potency in target-expressing cells, whereas the fluorophore-labeled product ADC suffers from a significant loss of activity, underscoring the impact of the label itself on the payload. Live-cell confocal microscopy reveals robust uptake of the clickable ADC, which reacts selectively in situ with a derivatized fluorescent label. Time-course trafficking studies show greater and more rapid net internalization of the ADCs than the parent antibody. More generally, the application of chemical biology tools to the study of ADCs should improve our understanding of how ADCs are processed in biological systems.

Protein biosynthesis and extracellular secretion are essential biological processes for therapeutic protein production in mammalian cells, which offer the capacity for correct folding and proper post-translational modifications. In this study, we have generated bispecific therapeutic fusion proteins in mammalian cells by combining a peptide and an antibody into a single open reading frame. A neutralizing peptide directed against interleukin-17A (IL17A) was genetically fused to the N termini of an anti-IL22 antibody, through either the light chain, the heavy chain, or both chains. Although the resulting fusion proteins bound and inhibited IL22 with the same affinity and potency as the unmodified anti-IL22 antibody, the peptide modality in the fusion scaffold was not active in the cell-based assay due to the N-terminal degradation. When a glutamine residue was introduced at the N terminus, which can be cyclized to form pyroglutamate in mammalian cells, the IL17A neutralization activity of the fusion protein was restored. Interestingly, the mass spectroscopic analysis of the purified fusion protein revealed an unexpected O-linked glycosylation modification at threonine 5 of the anti-IL17A peptide. The subsequent removal of this post-translational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein. These results provide direct experimental evidence that post-translational modifications during protein biosynthesis along secretory pathways play critical roles in determining the structure and function of therapeutic proteins produced by mammalian cells. The newly engineered peptide-antibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody-like molecule. Background: Protein biosynthesis and secretion are essential biological processes for therapeutic protein production. Results: Generating functional anti-IL17A and anti-IL22 peptide-antibody bispecific therapeutic proteins requires pyroglutamate addition and O-linked glycan removal. Conclusion: Post-translational modifications play critical roles in determining structure and function of therapeutic proteins. Significance: The peptide-antibody genetic fusion is promising for targeting multiple antigens in a single antibody-like molecule.

Protein modifications by intricate cellular machineries often redesign the structure and function of existing proteins to impact biological networks. Disulfide bond formation between cysteine (Cys) pairs is one of the most common modifications found in extracellularly-destined proteins, key to maintaining protein structure. Unpaired surface cysteines on secreted mammalian proteins are also frequently found disulfide-bonded with free Cys or glutathione (GSH) in circulation or culture, the mechanism for which remains unknown. Here we report that these so-called Cys-capping modifications take place outside mammalian cells, not in the endoplasmic reticulum (ER) where oxidoreductase-mediated protein disulfide formation occurs. Unpaired surface cysteines of extracellularly-arrived proteins such as antibodies are uncapped upon secretion before undergoing disulfide exchange with cystine or oxidized GSH in culture medium. This observation has led to a feasible way to selectively modify the nucleophilic thiol side-chain of cell-surface or extracellular proteins in live mammalian cells, by applying electrophiles with a chemical handle directly into culture medium. These findings provide potentially an effective approach for improving therapeutic conjugates and probing biological systems.

The market scale of China’s wind turbine manufacturing industry has grown immensely. Despite China still having a limited capacity in terms of technology innovation, the institutional support has promoted the technology capability development of the wind turbine manufacturing industry. This paper explores the driving forces underlying this development by reviewing the transition of the innovation modes and the dynamic interactions among the technology capability, innovation modes, market formation, and wind energy policy. The innovation mode in China began with imitative innovation, then transitioned to cooperative innovation, and has more recently set its sights on attaining truly indigenous innovation. Public policy serves as a key driving force for the evolution of innovation modes, as well as the development of the market. The policy focus has evolved in the following sequence: 1. building the foundation for technological innovation; 2. encouraging technology transfer; 3. enhancing local RD 4. enlarging the domestic market; and 5. cultivating an open environment for global competition and sustainable market development in China.

Large-scale transient expression in mammalian cells is a rapid protein production technology often used to shorten overall timelines for biotherapeutics drug discovery. In this study we demonstrate transient expression in a Chinese hamster ovary (CHO) host (ExpiCHO-S™) cell line capable of achieving high recombinant antibody expression titers, comparable to levels obtained using human embryonic kidney (HEK) 293 cells. For some antibodies, ExpiCHO-S™ cells generated protein materials with better titers and improved protein quality characteristics (i.e., less aggregation) than those from HEK293. Green fluorescent protein imaging data indicated that ExpiCHO-S™ displayed a delayed but prolonged transient protein expression process compared to HEK293. When therapeutic glycoproteins containing non-Fc N-linked glycans were expressed in transient ExpiCHO-S™, the glycan pattern was unexpectedly found to have few sialylated N-glycans, in contrast to glycans produced within a stable CHO expression system. To improve N-glycan sialylation in transient ExpiCHO-S™, we co-transfected galactosyltransferase and sialyltransferase genes along with the target genes, as well as supplemented the culture medium with glycan precursors. The authors have demonstrated that co-transfection of glycosyltransferases combined with medium addition of galactose and uridine led to increased sialylation content of N-glycans during transient ExpiCHO-S™ expression. These results have provided a scientific basis for developing a future transient CHO system with N-glycan compositions that are similar to those profiles obtained from stable CHO protein production systems. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2724, 2019.

Phosphodiesterase 11A (PDE11A) is the most recently identified family of phosphodiesterases (PDEs), the only known enzymes to break down cyclic nucleotides. The tissue expression profile of this dual specificity PDE is controversial, and little is understood of its biological function, particularly in the brain. We seek here to determine if PDE11A is expressed in the brain and to understand its function, using PDE11A −/− knockout (KO) mice. We show that PDE11A mRNA and protein are largely restricted to hippocampus CA1, subiculum, and the amygdalohippocampal area, with a two- to threefold enrichment in the ventral vs. dorsal hippocampus, equal distribution between cytosolic and membrane fractions, and increasing levels of protein expression from postnatal day 7 through adulthood. Interestingly, PDE11A KO mice show subtle psychiatric-disease–related deficits, including hyperactivity in an open field, increased sensitivity to the glutamate N -methyl- D -aspartate receptor antagonist MK-801, as well as deficits in social behaviors (social odor recognition memory and social avoidance). In addition, PDE11A KO mice show enlarged lateral ventricles and increased activity in CA1 (as per increased Arc mRNA), phenotypes associated with psychiatric disease. The increased sensitivity to MK-801 exhibited by PDE11A KO mice may be explained by the biochemical dysregulation observed around the glutamate α-amino-3-hydroxy-5-methyl-4-isozazolepropionic (AMPA) receptor, including decreased levels of phosphorylated-GluR1 at Ser845 and the prototypical transmembrane AMPA-receptor–associated proteins stargazin (γ2) and γ8. Together, our data provide convincing evidence that PDE11A expression is restricted in the brain but plays a significant role in regulating brain function.

The ‘war on cancer’ declared by President Nixon nearly 4 decades ago has fostered intensive research revealing the biological details and complexity of cancer. However, despite the global efforts in research, prevention and disease awareness, cancer incidence worldwide has continued to rise. Global demand for the commercial development of cancer medicines is growing, contributing partly to the rapid increase in health spending. One great challenge facing society is how to fund the implementation of cancer care in an increasingly expensive world under the current challenging economic circumstances. There is a growing dilemma regarding how much society is willing to pay for innovative drugs. This review finds that the reasons behind these developments can be attributed to several factors, including limited supply and rising demand for innovative drugs, significant cost increase in drug development and pricing expansion with growing fixed cost. Society must address these challenges by fairly defining and rewarding an appropriate value for innovative medicines, by determining their suitable portion in health expenditure and balancing healthcare resource prioritization.

Tissue specific amplification of glucocorticoid action through NADPH-dependent reduction of inactive glucocorticoid precursors by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) contributes to the development of visceral obesity, insulin resistance and Type 2 Diabetes. Hexose-6-phosphate dehydrogenase (H6PDH) is believed to supply NADPH for the reductase activity of 11beta-HSD1 in the lumen of the endoplasmic reticulum (ER), where the two enzymes are co-localized. We report here expression and purification of full-length and truncated N-terminal domain (NTD) of H6PDH in a mammalian expression system. Interestingly, both full-length H6PDH and the truncated NTD are secreted into the culture medium in the absence of 11beta-HSD1. Purified full-length H6PDH is a bi-functional enzyme with glucose-6-phosphate dehydrogenase (G6PDH) activity as well as 6-phosphogluconolactonase (6PGL) activity. Using co-immunoprecipitation experiments with purified H6PDH and 11beta-HSD1, and with cell lysates expressing H6PDH and 11beta-HSD1, we observe direct physical interaction between the two enzymes. We also show the modulation of 11beta-HSD1 directionality by H6PDH using overexpression and siRNA knockdown systems. The NTD retains the ability to interact with 11beta-HSD1 physically as well as modulate 11beta-HSD1 directionality indicating that the NTD of H6PDH is sufficient for the regulation of the 11beta-HSD1 activity.

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor-bound enzymes exhibit two essentially different catalytic-site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin.

The peptide-antibody (Ab) genetic fusion is a promising technology for targeting multiple antigens in a single Ab-like molecule. We have recently described generation and in vitro characterization of several such genetic fusions, using an interleukin (IL)-17A binding peptide and an anti-IL-22 Ab as a model system. In this study we assessed pharmacokinetic profiles of these model genetic fusions in mice. Specifically an IL-17A binding peptide was fused to either the heavy chain or both the heavy and the light chains of an anti-IL22 human IgG1 (referred to Compounds 1 or 2, respectively). Swiss Webster mice were given a single 10 mg/kg IV dose of Compound 1 or Compound 2 and serum concentrations were measured by a fused molecule immunoassay, in which IL-17A was used as a capture and anti-human IgG was used as a detector. In addition, serum samples were assayed using a total human IgG immunoassay. PK parameters were calculated by non-compartmental modeling. The two genetic fusions had similar PK profiles, with total body clearance of ~0.9-1.0 mL/h/kg, volume of distribution at steady-state of ~63-65 mL/kg, and elimination half-life of ~40 h. Our study provides the first characterization of the PK properties of peptide-Ab genetic fusions and suggests that although these genetic fusions appear to be eliminated faster than a typical Ab, the PK profile may be suitable for preclinical and clinical testing.

CD39 is a membrane-bound ecto-nucleoside triphosphate diphosphohydrolase that is involved in the regulation of purinergic signaling. It has been previously reported that N-linked glycosylation is essential for the surface localization of CD39 and for its cellular activity. Here we have addressed the roles of different stages of N-linked glycosylation on CD39's activity and surface expression by using various glycosylation inhibitors, glycosylation deficient CHO cells, and oligosaccharide removal enzymes. The results demonstrate that endoplasmic reticulum glycosylation is required for protein folding and essential for functional surface expression of CD39, while Golgi glycosylation is less important. The study has also shown that N-linked glycosylation of CD39 is dispensable for the activity after the protein is properly folded and targeted.

CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosphohydrolase family. The active site for native CD39 is located on the outer surface of the cellular plasma membrane; however, it is not yet known at what stage this enzyme becomes active along the secretory pathway to the plasma membrane. In this study, sucrose density fractionations performed on CD39-transfected COS-7 cell membranes suggest that CD39 activity resides primarily in the plasma membrane. Furthermore, we have created recombinant, soluble versions of CD39, one that is secreted and others that are retained in the endoplasmic reticulum, to demonstrate that CD39 is not active until it reaches the plasma membrane both in yeast and COS-7 cells. Moreover, the secreted active soluble CD39 in COS-7 cells is found to receive a higher degree of N-glycan addition than the inactive form retained intracellularly. When COS-7 cells were treated with tunicamycin to preventN-glycosylation, soluble CD39 was not detected in the extracellular medium and remained inactive intracellularly. Surface biotinylation analysis also revealed that surface-expressed wild type CD39 receives a higher degree of N-glycosylation than intracellular forms and that inhibition of N-glycosylation prevents its plasma membrane localization. In addition, both intact and digitonin-permeablized COS-7 cells transfected with CD39 possess similar ecto-ATPase activities, further supporting the conclusion that only surface-expressed CD39 is enzymatically active. All of these data suggest that intracellular CD39 is inactive and that only a fully glycosylated CD39 has apyrase activity and is localized at the cell surface.

CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. By using a two-hybrid screen, we found that an activator subunit (Vma13p) of yeast vacuolar H(+)-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation. Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Delta mutant was drastically increased compared with that of Ynd1p in VMA13 cells. A similar increase in the apyrase activity of Ynd1p was found in a vma1Delta mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes. However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels of Ynd1p activity, demonstrating that the presence of Vma13p rather than the function of V-ATPase in the membrane represses Ynd1p activity. These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from Escherichia coli is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg 2+ and Mn 2+ dependent. However, BCTD possesses similar GTPase and ATPase activities at 37°C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10°C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.

The antibacterial peptide toxin colicin V uses a dedicated signal sequence-independent system for its secretion in Escherichia coli and requires the products of three genes, cvaA, cvaB, and tolC. As a member of the membrane fusion protein family, CvaA is supposed to form a bridge that connects the inner and outer membranes via interaction with CvaB and TolC, respectively. In this study, we investigated the possible interaction of these proteins. When CvaA or CvaB was absent, the corresponding amount of CvaB or CvaA, respectively, was decreased, and the amounts of both proteins were reduced when TolC was depleted. Translational lacZ fusions showed that TolC did not affect the synthesis of either CvaA-beta-galactosidase or CvaB-beta-galactosidase, and CvaA or CvaB did not affect the synthesis of CvaB-beta-galactosidase or CvaA-beta-galactosidase, respectively. However, the stabilities of CvaA and CvaB proteins were affected by the absence of one another and by that of TolC. The instability of CvaA was more severe in TolC-depleted cells than in CvaB-depleted cells. On the other hand, CvaB was less stable in the absence of CvaA than in the absence of TolC. In addition, using a cross-linking reagent, we showed that CvaA directly interacts with both CvaB and TolC proteins. Taken together, these data support the hypothesized structural role of CvaA in connecting CvaB and TolC.

While myriad molecular formats for bispecific antibodies have been examined to date, the simplest structures are often based on the scFv. Issues with stability and manufacturability in scFv-based bispecific molecules, however, have been a significant hindrance to their development, particularly for high-concentration, stable formulations that allow subcutaneous delivery. Our aim was to generate a tetravalent bispecific molecule targeting two inflammatory mediators for synergistic immune modulation. We focused on an scFv-Fc-scFv format, with a flexible (A4T)3 linker coupling an additional scFv to the C-terminus of an scFv-Fc. While one of the lead scFvs isolated directly from a naive library was well-behaved and sufficiently potent, the parental anti-CXCL13 scFv 3B4 required optimization for affinity, stability, and cynomolgus ortholog cross-reactivity. To achieve this, we eschewed framework-based stabilizing mutations in favor of complementarity-determining region (CDR) mutagenesis and re-selection for simultaneous improvements in both affinity and thermal stability. Phage-displayed 3B4 CDR-mutant libraries were used in an aggressive “hammer-hug” selection strategy that incorporated thermal challenge, functional, and biophysical screening. This approach identified leads with improved stability and >18-fold, and 4,100-fold higher affinity for both human and cynomolgus CXCL13, respectively. Improvements were exclusively mediated through only 4 mutations in VL-CDR3. Lead scFvs were reformatted into scFv-Fc-scFvs and their biophysical properties ranked. Our final candidate could be formulated in a standard biopharmaceutical platform buffer at 100 mg/ml with

Glycosylation is one of the most complex post-translation modifications, commonly found in many cell surface and secreted eukaryotic proteins. 1-2% of the human transcriptome encodes proteins that link to glycosylation. Many protein-based biotherapeutics approved or in clinical trials are glycoproteins. The oligosaccharides covalently attached to therapeutic glycoproteins pose biological benefits as well as manufacturing challenges. The present chapter reviews the structure and function of glycosylation, glycoform patterns observed for the biotherapeutic proteins produced by various host systems, and analytic methods for the characterization of glycoforms. Recent advances in utilizing glycosylation as a strategy to improve biotherapeutics properties are also discussed.

Biotherapeutics drugs, such as antibodies, Fc-like fusion proteins, and therapeutic replacement enzymes, constitute the most rapidly growing drug class, and have become a major clinical success of human therapeutics over the past decade. These therapeutics of large-molecule have revolutionized the treatment of a variety of diseases in areas such as oncology, inflammatory and autoimmune diseases, hemophilia, cardiovascular disease, infectious diseases, and rare genetic diseases. In comparison with small-molecule therapeutics, biotherapeutics have a higher approval success rate and a similar development phase length. Physician and patients have accepted biotherapeutics drugs even though most of these products are administrated via injection. Many new biotherapeutics candidates are filling the pre-clinical and clinical pipelines of major Biopharmaceutical companies. The aim of this chapter is to provide a review on recent advances in Biotherapeutics drug discovery and development. Action mechanisms, tools for biotherapeutics generation, design processes, issues like safety and side effects, will be described. In addition, pharmacoeconomics and strategies to provide affordable biotherapeutics drugs will be discussed.

Many secreted or cell surface proteins are post-translationally modified by carbohydrate chains which are a primary source of heterogeneity. The Lec1 mutant, which is defective in Golgi N-acetylglucosaminyltransferase I (GnTI) activity, produces relatively homogeneous Man(5) GlcNAc(2) glycan modifications, and is widely used for various applications. To facilitate the investigation of GnTI, its Man5 glycan endproduct, and the impact of Man5 on effector function, the present study has established several novel Lec1 mutants in dhfr(-) CHO-DUKX cells through chemical mutagenesis and lectin selection. A total of nine clonal lines exhibiting the Lec1-phenotype are characterized, six of which harbor non-sense mutations leading to a truncated GnTI, and three (R415K, D291N, and P138L) of which are novel loss-of-function sense mutations. Analysis of the rabbit GnTI structure (Unligil et al., 2000) indicates that D291 is the proposed catalytic base and R415 is a crucial residue in forming the substrate binding pocket, whereas P138 is key to maintaining two β strands in proximity to the substrate binding pocket. Computational modeling reveals that the oligomannose glycan backbone of a glycoprotein (the acceptor substrate) fits nicely into the unoccupied channel of the substrate binding pocket partly through hydrogen bonding with R415 and D291. This finding is consistent with the ordered sequential Bi Bi kinetic mechanism suggested for GnTI, in which binding of UDP-GlcNAc (the donor substrate)/Mn(2+) induces conformational changes that promote acceptor binding. When an anti-human CD20 antibody protein is stably expressed in one CHO-DUKX-Lec1 line, it is confirmed that N-glycans are predominantly Man(5) GlcNAc(2) and they do not contain an α1,6-fucose linked to the innermost GlcNAc. Furthermore, this Man(5) GlcNAc(2) modified antibody exhibits a significantly increased ADCC activity than the wild-type protein, while displaying a lower CDC activity. The data support the hypothesis that modulating GnTI activity can influence antibody effector functions for proteins with an IgG1 immunoglobulin Fc domain.

Advances in genomics and proteomics have generated the needs for the efficient identification of key residues for structure and function of target proteins. Here we report the utilization of a new residue-screening approach, which combines a mammalian high-throughput transient expression system with a PCR-based expression cassette, for the study of the post-translational modification. Applying this approach results in a quick identification of essential N-glycosylation sites of a heavily glycosylated neuroglycoprotein Lingo-1, which are sufficient for the support of its surface expression. These key N-glycosylated sites uniquely locate on the concave surface of the elongated arc-shape structure of the leucine-rich repeat domain. The swift residue-screening approach may provide a new strategy for structural and functional analysis.

CD39 is a prototype member of the ecto-nucleoside triphosphate diphosphohydrolase family that hydrolyzes extracellular nucleoside diphosphates and triphosphates in the presence of divalent cations. Here, the expression, purification and crystallization of the ecto-enzymatic domain of rat CD39, sCD39, are described. The 67 kDa secreted soluble glycoprotein was recombinantly overexpressed in a glycosylation mutant CHO line, Lec.3.2.8.1, and purified from conditioned media. Diffraction-quality crystals of sCD39 were produced by the vapor-diffusion method using PEG 3350 and ammonium dihydrogen phosphate as precipitants. The enzyme crystallized in a primitive trigonal form in space group P3(2), with unit-cell parameters a = b = 118.1, c = 81.6 A and with two sCD39 copies in the asymmetric unit. Several low- to medium-resolution diffraction data sets were collected using an in-house X-ray source. Analysis of the intensity statistics showed that the crystals were invariably merohedrally twinned with a high twin fraction. For initial phasing, a molecular-replacement search was performed against the complete 3.2 A data set using a maximum-likelihood molecular-replacement method as implemented in Phaser. The initial model of the two sCD39 monomers was placed into the P3(2) lattice and rigid-body refined and position-minimized with PHENIX.