Engineered CHO cells as a novel AAV production platform for gene therapy delivery.

The Herpes simplex virus (HSV)-based platform for production of recombinant adeno-associated viral vectors (rAAVs) yields higher titers and increased percentage of full capsids when compared to the triple transient transfection (TTT) method. However, this platform currently faces two major challenges. The first challenge is the reliance on commercial media, sometimes supplemented with serum, leading to costly manufacturing and a high risk for introduction of adventitious agents. The second challenge is that the production of HSV-1 relies on adherent complementing Vero cells (V27), making it difficult to scale up. We engineered serum-free-adapted CHO cells expressing key HSV-1 entry receptors, HVEM and/or Nectin-1 to address the first challenge. Using high-throughput cloning methods, we successfully selected a HVEM receptor-expressing clone (CHO-HV-C1) that yields 1.62 × 109, 2.51 × 109, and 4.07 × 109 viral genome copies/mL with rAAV6.2-GFP, rAAV8-GFP, and rAAV9-GFP vectors respectively, within 24 h post rHSV-1 co-infection. Moreover, CHO-HV-C1-derived rAAVs had comparable in vitro transduction, infectivity, and biodistribution titers to those produced by TTT. The second challenge was addressed via engineering CHO-HV-C1 cells to express HSV-1 CP27. These cells successfully produced rHSV-1 vectors, but with significantly lower titers than V27 cells. Taken together, the CHO/HSV system provides a novel, scalable, reduced cost, serum-free AAV manufacturing platform.

Stochastic assembly of biomacromolecular complexes: impact and implications on charge interpretation in native mass spectrometry.

Native mass spectrometry is a potent method for characterizing biomacromolecular assemblies. A critical aspect to extracting accurate mass information is the correct inference of the ion ensemble charge states. While a variety of experimental strategies and algorithms have been developed to facilitate this, virtually all approaches rely on the implicit assumption that any peaks in a native mass spectrum can be directly attributed to an underlying charge state distribution. Here, we demonstrate that this paradigm breaks down for several types of macromolecular protein complexes due to the intrinsic heterogeneity induced by the stochastic nature of their assembly. Utilizing several protein assemblies of adeno-associated virus capsids and ferritin, we demonstrate that these particles can produce a variety of unexpected spectral appearances, some of which appear superficially similar to a resolved charge state distribution. When interpreted using conventional charge inference strategies, these distorted spectra can lead to substantial errors in the calculated mass (up to ∼5%). We provide a novel analytical framework to interpret and extract mass information from these spectra by combining high-resolution native mass spectrometry, single particle Orbitrap-based charge detection mass spectrometry, and sophisticated spectral simulations based on a stochastic assembly model. We uncover that these mass spectra are extremely sensitive to not only mass heterogeneity within the subunits, but also to the magnitude and width of their charge state distributions. As we postulate that many protein complexes assemble stochastically, this framework provides a generalizable solution, further extending the usability of native mass spectrometry in the characterization of biomacromolecular assemblies.

Multi-Attribute Monitoring Method for Process Development of Engineered Antibody for Site-Specific Conjugation.

Antibody drug conjugates, a class of biotherapeutic proteins, have been extensively developed in recent years, resulting in new approvals and improved standard of care for cancer patients. Among the numerous strategies of conjugating cytotoxic payloads to monoclonal antibodies, insertion of a cysteine residue achieves a tightly controlled, site-specific drug to antibody ratio. Tailored analytical tools are required to direct the development of processes capable of manufacturing novel antibody scaffolds with the desired product quality. Here, we describe the development of a 12 min, mass-spectrometry-based method capable of monitoring four distinct quality attributes simultaneously: variations in the thiol state of the inserted cysteines, N-linked glycosylation, reduction of interchain disulfide bonds, and polypeptide fragmentation. This method provides new insight into the properties of the antibody intermediate and associated manufacturing processes. Oxidized thiol states are formed within the bioreactor, of which a variant containing an additional disulfide bond was produced and remained relatively constant throughout the fed-batch process; reduced thiol variants were introduced upon harvest. Nearly 20 percent of N-linked glycans contained sialic acid, substantially higher than anticipated for wildtype IgG1. Lastly, previously unreported polypeptide fragmentation sites were identified in the C239i constant domain, and the relationship between fragmentation and glycoform were explored. This work illustrates the utility of applying a high-throughput liquid chromatography-mass spectrometry multi-attribute monitoring method to support the development of engineered antibody scaffolds.

Mechanistic insights into the rational design of masked antibodies.

Although monoclonal antibodies have greatly improved cancer therapy, they can trigger side effects due to on-target, off-tumor toxicity. Over the past decade, strategies have emerged to successfully mask the antigen-binding site of antibodies, such that they are only activated at the relevant site, for example, after proteolytic cleavage. However, the methods for designing an ideal affinity-based mask and what parameters are important are not yet well understood. Here, we undertook mechanistic studies using three masks with different properties and identified four critical factors: binding site and affinity, as well as association and dissociation rate constants, which also played an important role. HDX-MS was used to identify the location of binding sites on the antibody, which were subsequently validated by obtaining a high-resolution crystal structure for one of the mask-antibody complexes. These findings will inform future designs of optimal affinity-based masks for antibodies and other therapeutic proteins.

A chemoenzymatic strategy for site-selective functionalization of native peptides and proteins.

The emergence of new therapeutic modalities requires complementary tools for their efficient syntheses. Availability of methodologies for site-selective modification of biomolecules remains a long-standing challenge, given the inherent complexity and the presence of repeating residues that bear functional groups with similar reactivity profiles. We describe a bioconjugation strategy for modification of native peptides relying on high site selectivity conveyed by enzymes. We engineered penicillin G acylases to distinguish among free amino moieties of insulin (two at amino termini and an internal lysine) and manipulate cleavable phenylacetamide groups in a programmable manner to form protected insulin derivatives. This enables selective and specific chemical ligation to synthesize homogeneous bioconjugates, improving yield and purity compared to the existing methods, and generally opens avenues in the functionalization of native proteins to access biological probes or drugs.

A kinase-cGAS cascade to synthesize a therapeutic STING activator.

The introduction of molecular complexity in an atom- and step-efficient manner remains an outstanding goal in modern synthetic chemistry. Artificial biosynthetic pathways are uniquely able to address this challenge by using enzymes to carry out multiple synthetic steps simultaneously or in a one-pot sequence1-3. Conducting biosynthesis ex vivo further broadens its applicability by avoiding cross-talk with cellular metabolism and enabling the redesign of key biosynthetic pathways through the use of non-natural cofactors and synthetic reagents4,5. Here we describe the discovery and construction of an enzymatic cascade to MK-1454, a highly potent stimulator of interferon genes (STING) activator under study as an immuno-oncology therapeutic6,7 (ClinicalTrials.gov study NCT04220866 ). From two non-natural nucleotide monothiophosphates, MK-1454 is assembled diastereoselectively in a one-pot cascade, in which two thiotriphosphate nucleotides are simultaneously generated biocatalytically, followed by coupling and cyclization catalysed by an engineered animal cyclic guanosine-adenosine synthase (cGAS). For the thiotriphosphate synthesis, three kinase enzymes were engineered to develop a non-natural cofactor recycling system in which one thiotriphosphate serves as a cofactor in its own synthesis. This study demonstrates the substantial capacity that currently exists to use biosynthetic approaches to discover and manufacture complex, non-natural molecules.

Antisense oligonucleotide activity in tumour cells is influenced by intracellular LBPA distribution and extracellular vesicle recycling.

Next generation modified antisense oligonucleotides (ASOs) are commercially approved new therapeutic modalities, yet poor productive uptake and endosomal entrapment in tumour cells limit their broad application. Here we compare intracellular traffic of anti KRAS antisense oligonucleotide (AZD4785) in tumour cell lines PC9 and LK2, with good and poor productive uptake, respectively. We find that the majority of AZD4785 is rapidly delivered to CD63+late endosomes (LE) in both cell lines. Importantly, lysobisphosphatidic acid (LBPA) that triggers ASO LE escape is presented in CD63+LE in PC9 but not in LK2 cells. Moreover, both cell lines recycle AZD4785 in extracellular vesicles (EVs); however, AZD4785 quantification by advanced mass spectrometry and proteomic analysis reveals that LK2 recycles more AZD4785 and RNA-binding proteins. Finally, stimulating LBPA intracellular production or blocking EV recycling enhances AZD4785 activity in LK2 but not in PC9 cells thus offering a possible strategy to enhance ASO potency in tumour cells with poor productive uptake of ASOs.

Interconversion of Unexpected Thiol States Affects the Stability, Structure, and Dynamics of Antibody Engineered for Site-Specific Conjugation.

Antibody-drug conjugates have become one of the most actively developed classes of drugs in recent years. Their great potential comes from combining the strengths of large and small molecule therapeutics: the exquisite specificity of antibodies and the highly potent nature of cytotoxic compounds. More recently, the approach of engineering antibody-drug conjugate scaffolds to achieve highly controlled drug to antibody ratios has focused on substituting or inserting cysteines to facilitate site-specific conjugation. Herein, we characterize an antibody scaffold engineered with an inserted cysteine that formed an unexpected disulfide bridge during manufacture. A combination of mass spectrometry and biophysical techniques have been used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody intermediate. This quantitative and structurally resolved model of the local and global changes in structure and dynamics associated with the engineering and subsequent disulfide-bonded variant can assist future engineering strategies.

Hormone therapy use and the risk of acute kidney injury in patients with prostate cancer: a population-based cohort study.

BACKGROUND: Hormone therapy is widely used in prostate cancer. However, studies have raised concerns that hormone therapy, particularly the use of gonadotropin-releasing hormone agonists, could increase the risk of acute kidney injury. METHODS: Men newly diagnosed with non-metastatic prostate cancer, from 2012 to 2017, were identified from the Scottish Cancer Registry. A matched comparison cohort of prostate cancer-free men was also identified. Hormone therapy use was determined from the Prescribing Information System in Scotland. The primary outcome was hospitalisations with acute kidney injury taken from Scottish hospital records (SMR01) up to June 2019. Time-dependent Cox regression models were used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for acute kidney injury by hormone therapy use. RESULTS: The prostate cancer cohort contained 10,751 patients followed for 41,997 person years, during which there were 618 hospitalisations with acute kidney injury. Prostate cancer patients had higher rates of acute kidney injury compared with cancer-free controls (adjusted HR = 1.47 95% CI 1.29, 1.69). However, prostate cancer patients currently using hormone therapy (adjusted HR = 1.14 95% CI 0.92, 1.41), including gonadotropin-releasing hormone (GnRH) agonists (adjusted HR = 1.13 95% CI 0.90, 1.40), did not appear to have a marked increase in acute kidney injury compared with prostate cancer patients not using hormone therapy after adjusting for potential confounders. CONCLUSIONS: In our cohort, there was little evidence that gonadotropin-releasing hormone agonists were associated with marked increases in acute kidney injury.

An in vivo platform to select and evolve aggregation-resistant proteins.

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve 'manufacturable' biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.

Biocatalysis in drug discovery and development.

Enzyme catalysis, enabled by advances in protein engineering and directed evolution, is beginning to transform chemical synthesis in the pharmaceutical industry. This review presents recent examples of the creative use of biocatalysis to enable drug discovery and development. We illustrate how increased access to novel biotransformations and the rise of cascade biocatalysis allowed fundamentally new syntheses of novel medicines, representing progress toward more sustainable pharmaceutical manufacturing. Finally, we describe the opportunities and challenges the industry must address to ensure the reduction to practice of biotechnological innovations to develop new therapies in a faster, more economical, and environmentally benign way.

Association of soluble ST2 with all-cause and cardiovascular mortality in renal transplant recipients: a single-centre cohort study.

BACKGROUND: Soluble ST2 is a novel biomarker of myocardial fibrosis with an established role in prognostication of patients with heart failure. Its role in cardiovascular risk prediction for renal transplant recipients has not been investigated despite promising results for ST2 in other populations with renal disease. METHODS: In this prospective cohort study, 367 renal transplant recipients were followed up for a median of 16.2 years to investigate the association of soluble ST2 concentration with all-cause mortality. Cardiovascular mortality and major adverse cardiovascular events were secondary outcomes. Cox regression models were used to calculate hazard ratios and 95% confidence intervals for ST2 before and after adjustments. ST2 concentration was analysed both as a continuous variable and following categorisation according to the recommended cut-point of 35 ng/ml. RESULTS: A twofold higher ST2 concentration was associated with a 36% increased risk of all-cause mortality after adjustment for conventional cardiovascular risk factors and high-sensitivity C-reactive protein (adjusted hazard ratio 1.36; 95% confidence interval 1.06-1.75; p = 0.016). Associations with ST2 concentration were similar for cardiovascular events (adjusted hazard ratio 1.31; 95% confidence interval 1.00-1.73; p = 0.054), but were stronger for cardiovascular mortality (adjusted hazard ratio 1.61; 95% confidence interval 1.07-2.41; p = 0.022). Addition of ST2 to risk prediction models for mortality and cardiovascular events failed to improve their predictive accuracy. CONCLUSIONS: ST2 is associated with, but does not improve prediction of, adverse outcomes in renal transplant recipients.

Trends in Salmonella serovars and antimicrobial resistance in pigs and poultry in Northern Ireland between 1997 and 2016.

BACKGROUND: In the EU, salmonellosis is the second most commonly reported zoonosis. This pattern is reflected in Northern Ireland. Historically, foodborne salmonellosis has largely been attributed to the consumption of poultry products, and as such a number of legislative measures have been introduced by the EC. These policies focus mainly on five target Salmonella serovars. METHODS: Here the authors present a descriptive analysis of 20 years of data from the Northern Ireland National Reference Laboratory for Salmonella. RESULTS: The study's results show, for poultry submissions, a large decrease in the detection of four of the five targeted Salmonella serovars over the study period, with the fifth serovar undetected throughout the study. Additionally, there was an increase in the detection of a number of other non-regulated serovars. In pigs, S Typhimurium, which is among the most common causes of human salmonellosis, was the most commonly isolated serovar. When comparing levels of antimicrobial resistance in S Typhimurium between livestock groups, the authors found a decrease over time in poultry, but an increase in pigs, highlighting the potential significance of pigs in addressing public health concerns. CONCLUSION: The authors conclude that continued surveillance is important in the assessment of control measures at a national and transnational scale.

Mass Activated Droplet Sorting (MADS) Enables High-Throughput Screening of Enzymatic Reactions at Nanoliter Scale.

Microfluidic droplet sorting enables the high-throughput screening and selection of water-in-oil microreactors at speeds and volumes unparalleled by traditional well-plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high-throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI-MS). Droplets are split, one portion is analyzed by ESI-MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s-1 is achieved with 98 % accuracy using a self-correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label-free ESI-MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.

Design of an in vitro biocatalytic cascade for the manufacture of islatravir.

Enzyme-catalyzed reactions have begun to transform pharmaceutical manufacturing, offering levels of selectivity and tunability that can dramatically improve chemical synthesis. Combining enzymatic reactions into multistep biocatalytic cascades brings additional benefits. Cascades avoid the waste generated by purification of intermediates. They also allow reactions to be linked together to overcome an unfavorable equilibrium or avoid the accumulation of unstable or inhibitory intermediates. We report an in vitro biocatalytic cascade synthesis of the investigational HIV treatment islatravir. Five enzymes were engineered through directed evolution to act on non-natural substrates. These were combined with four auxiliary enzymes to construct islatravir from simple building blocks in a three-step biocatalytic cascade. The overall synthesis requires fewer than half the number of steps of the previously reported routes.

The antiquity of Nullarbor speleothems and implications for karst palaeoclimate archives.

Speleothems represent important archives of terrestrial climate variation that host a variety of proxy signals and are also highly amenable to radiometric age determination. Although speleothems have been forming on Earth for at least 400 million years, most studies rely upon the U-Th chronometer which extends only to the mid Pleistocene, leaving important questions over their longer-term preservation potential. To date, older records, exploiting the advantages of the U-Pb chronometer, remain fragmentary 'snapshots in time'. Here we demonstrate the viability of speleothems as deep time climate archives by showing that a vast system of shallow caves beneath the arid Nullarbor plain of southern Australia, the world's largest exposed karst terrain, formed largely within the Pliocene epoch, with a median age of 4.2 Ma, and that, in these caves, even the most delicate formations date from this time. The long-term preservation of regional-scale cave networks such as this demonstrates that abundant speleothem archives do survive to permit the reconstruction of climates and environments for much older parts of Earth history than the ~600 ka period to which most previous studies have been limited.

Hemoprotein-Catalyzed Cyclopropanation En Route to the Chiral Cyclopropanol Fragment of Grazoprevir.

Reactions that were once the exclusive province of synthetic catalysts can increasingly be addressed using biocatalysis. Through discovery of unnatural enzyme reactions, biochemists have significantly expanded the reach of enzymatic catalysis to include carbene transfer chemistries including olefin cyclopropanation. Here we describe hemoprotein cyclopropanation catalysts derived from thermophilic bacterial globins that react with diazoacetone and an unactivated olefin substrate to furnish a cyclopropyl ketone, a previously unreported reaction for enzyme catalysts. We further demonstrate that the resulting cyclopropyl ketone can be converted to a key cyclopropanol intermediate that occurs en route to the anti-hepatitis C drug grazoprevir.

Cardiovascular risk in renal transplant recipients.

Successful kidney transplantation offers patients with end-stage renal disease the greatest likelihood of survival. However, cardiovascular disease poses a major threat to both graft and patient survival in this cohort. Transplant recipients are unique in their accumulation of a wide range of traditional and non-traditional cardiovascular risk factors. Hypertension, diabetes, dyslipidaemia and obesity are highly prevalent in patients with end-stage renal disease. These risk factors persist following transplantation and are often exacerbated by the drugs used for immunosuppression in organ transplantation. Additional transplant-specific factors such as poor graft function and proteinuria are also associated with increased cardiovascular risk. However, these transplant-related factors remain unaccounted for in current cardiovascular risk prediction models, making it challenging to identify transplant recipients with highest risk. With few interventional trials in this area specific to transplant recipients, strategies to reduce cardiovascular risk are largely extrapolated from other populations. Aggressive management of traditional cardiovascular risk factors remains the cornerstone of prevention, though there is also a potential role for selecting immunosuppression regimens to minimise additional cardiovascular injury.

Synthesis of Grazoprevir, a Potent NS3/4a Protease Inhibitor for the Treatment of Hepatitis C Virus.

An efficient synthesis of grazoprevir is reported. Starting from four readily available building blocks, grazoprevir is prepared in 51% overall yield and >99.9% purity for pharmaceutical use.