Anticipatory regulation for pandemic responses: are we there yet?

Can drug and vaccine regulatory agencies leverage their experience during the coronavirus disease 2019 (COVID-19) pandemic to advance from reactive regulation to adaptive regulation and beyond to anticipatory regulation to prevent or curb future pandemics?

Amino acid degradation pathway inhibitory by-products trigger apoptosis in CHO cells.

Chinese hamster ovary (CHO) cells are widely used to produce complex biopharmaceuticals. Improving their productivity is necessary to fulfill the growing demand for such products. One way to enhance productivity is by cultivating cells at high densities, but inhibitory by-products, such as metabolite derivatives from amino acid degradation, can hinder achieving high cell densities. This research examines the impact of these inhibitory by-products on high-density cultures. We cultured X1 and X2 CHO cell lines in a small-scale semi-perfusion system and introduced a mix of inhibitory by-products on day 10. The X1 and X2 cell lines were chosen for their varied responses to the by-products; X2 was susceptible, while X1 survived. Proteomics revealed that the X2 cell line presented changes in the proteins linked to apoptosis regulation, cell building block synthesis, cell growth, DNA repair, and energy metabolism. We later used the AB cell line, an apoptosis-resistant cell line, to validate the results. AB behaved similar to X1 under stress. We confirmed the activation of apoptosis in X2 using a caspase assay. This research provides insights into the mechanisms of cell death triggered by inhibitory by-products and can guide the optimization of CHO cell culture for biopharmaceutical manufacturing.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

Are Manufacturing Patents to Blame for Biosimilar Market Launch Delays?

OBJECTIVES: Biosimilar market launch delays are likely costing healthcare systems billions of dollars and preventing patients accessing affordable biologic therapies sooner. Many claim these delays are mostly caused by originator biologics' large patent portfolios asserted during litigation against biosimilar developers, particularly that the manufacturing patents filed after the originator is approved is an important driver of these delays. Our objective was to investigate the accuracy of these claims. METHODS: We reviewed US Court document submissions for litigation data, including the details of patents asserted against biosimilar owners, and collated biosimilar market launch dates from publicly available databases. RESULTS: We find that, although approximately half of all patents asserted in litigation were manufacturing patents, a greater proportion of composition, active pharmaceutical ingredient, and treatment patents are associated with longer market launch delays, whereas a greater proportion of manufacturing patents are associated with shorter market launch delays. CONCLUSIONS: Our results suggest that manufacturing patents were having less of an impact on market launch delays than other types of patents. Our findings have implications for both biosimilar and originator developers, as well as patent policy and its association with healthcare accessibility.

Long-term safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2 in adults aged 18-55 years or ≥56 years: 12-month results from a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: We previously demonstrated the safety and immunogenicity of an MF59-adjuvanted COVID-19 vaccine based on the SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a molecular clamp using HIV-1 glycoprotein 41 sequences. Here, we describe 12-month results in adults aged 18-55 years and ≥56 years. METHODS: Phase 1, double-blind, placebo-controlled trial conducted in Australia (July 2020-December 2021; ClinicalTrials.govNCT04495933; active, not recruiting). Healthy adults (Part 1: 18-55 years; Part 2: ≥56 years) received two doses of placebo, 5 µg, 15 µg, or 45 µg vaccine, or one 45 µg dose of vaccine followed by placebo (Part 1 only), 28 days apart (n = 216; 24 per group). Safety, humoral immunogenicity (including against virus variants), and cellular immunogenicity were assessed to day 394 (12 months after second dose). Effects of subsequent COVID-19 vaccination on humoral responses were examined. FINDINGS: All two-dose vaccine regimens were well tolerated and elicited strong antigen-specific and neutralising humoral responses, and CD4+ T-cell responses, by day 43 in younger and older adults, although cellular responses were lower in older adults. Humoral responses waned by day 209 but were boosted in those receiving authorised vaccines. Neutralising activity against Delta and Omicron variants was present but lower than against the Wuhan strain. Cross-reactivity in HIV diagnostic tests declined over time but remained detectable in most participants. INTERPRETATION: The SARS-CoV-2 molecular clamp vaccine is well tolerated and evokes robust immune responses in adults of all ages. Although the HIV glycoprotein 41-based molecular clamp is not being progressed, the clamp concept represents a viable platform for vaccine development. FUNDING: This study was funded by the Coalition for Epidemic Preparedness Innovations, the National Health and Medical Research Council of Australia, and the Queensland Government.

mRNA vaccine quality analysis using RNA sequencing.

The success of mRNA vaccines has been realised, in part, by advances in manufacturing that enabled billions of doses to be produced at sufficient quality and safety. However, mRNA vaccines must be rigorously analysed to measure their integrity and detect contaminants that reduce their effectiveness and induce side-effects. Currently, mRNA vaccines and therapies are analysed using a range of time-consuming and costly methods. Here we describe a streamlined method to analyse mRNA vaccines and therapies using long-read nanopore sequencing. Compared to other industry-standard techniques, VAX-seq can comprehensively measure key mRNA vaccine quality attributes, including sequence, length, integrity, and purity. We also show how direct RNA sequencing can analyse mRNA chemistry, including the detection of nucleoside modifications. To support this approach, we provide supporting software to automatically report on mRNA and plasmid template quality and integrity. Given these advantages, we anticipate that RNA sequencing methods, such as VAX-seq, will become central to the development and manufacture of mRNA drugs.

A nanobody recognizes a unique conserved epitope and potently neutralizes SARS-CoV-2 omicron variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) Omicron variant sub-lineages spread rapidly worldwide, mostly due to their immune-evasive properties. This has put a significant part of the population at risk for severe disease and underscores the need for effective anti-SARS-CoV-2 agents against emergent strains in vulnerable patients. Camelid nanobodies are attractive therapeutic candidates due to their high stability, ease of large-scale production, and potential for delivery via inhalation. Here, we characterize the receptor binding domain (RBD)-specific nanobody W25 and show superior neutralization activity toward Omicron sub-lineages in comparison to all other SARS-CoV2 variants. Structure analysis of W25 in complex with the SARS-CoV2 spike glycoprotein shows that W25 engages an RBD epitope not covered by any of the antibodies previously approved for emergency use. In vivo evaluation of W25 prophylactic and therapeutic treatments across multiple SARS-CoV-2 variant infection models, together with W25 biodistribution analysis in mice, demonstrates favorable pre-clinical properties. Together, these data endorse W25 for further clinical development.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

SARS-CoV-2 drives NLRP3 inflammasome activation in human microglia through spike protein.

Coronavirus disease-2019 (COVID-19) is primarily a respiratory disease, however, an increasing number of reports indicate that SARS-CoV-2 infection can also cause severe neurological manifestations, including precipitating cases of probable Parkinson's disease. As microglial NLRP3 inflammasome activation is a major driver of neurodegeneration, here we interrogated whether SARS-CoV-2 can promote microglial NLRP3 inflammasome activation. Using SARS-CoV-2 infection of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) as a COVID-19 pre-clinical model, we established the presence of virus in the brain together with microglial activation and NLRP3 inflammasome upregulation in comparison to uninfected mice. Next, utilising a model of human monocyte-derived microglia, we identified that SARS-CoV-2 isolates can bind and enter human microglia in the absence of viral replication. This interaction of virus and microglia directly induced robust inflammasome activation, even in the absence of another priming signal. Mechanistically, we demonstrated that purified SARS-CoV-2 spike glycoprotein activated the NLRP3 inflammasome in LPS-primed microglia, in a ACE2-dependent manner. Spike protein also could prime the inflammasome in microglia through NF-κB signalling, allowing for activation through either ATP, nigericin or α-synuclein. Notably, SARS-CoV-2 and spike protein-mediated microglial inflammasome activation was significantly enhanced in the presence of α-synuclein fibrils and was entirely ablated by NLRP3-inhibition. Finally, we demonstrate SARS-CoV-2 infected hACE2 mice treated orally post-infection with the NLRP3 inhibitory drug MCC950, have significantly reduced microglial inflammasome activation, and increased survival in comparison with untreated SARS-CoV-2 infected mice. These results support a possible mechanism of microglial innate immune activation by SARS-CoV-2, which could explain the increased vulnerability to developing neurological symptoms akin to Parkinson's disease in COVID-19 infected individuals, and a potential therapeutic avenue for intervention.

Strategies for developing a recombinant butyrylcholinesterase medical countermeasure for Organophosphorus poisoning.

Organophosphorus nerve agents represent a serious chemical threat due to their ease of production and scale of impact. The recent use of the nerve agent Novichok has re-emphasised the need for broad-spectrum medical countermeasures (MCMs) to these agents. However, current MCMs are limited. Plasma derived human butyrylcholinesterase (huBChE) is a promising novel bioscavenger MCM strategy, but is prohibitively expensive to isolate from human plasma at scale. Efforts to produce recombinant huBChE (rBChE) in various protein expression platforms have failed to achieve key critical attributes of huBChE such as circulatory half-life. These proteins often lack critical features such as tetrameric structure and requisite post-translational modifications. This review evaluates previous attempts to generate rBChE and assesses recent advances in mammalian cell expression and protein engineering strategies that could be deployed to achieve the required half-life and yield for a viable rBChE MCM. This includes the addition of a proline-rich attachment domain, fusion proteins, post translational modifications, expression system selection and optimised downstream processes. Whilst challenges remain, a combinatorial application of these strategies demonstrates potential as a technically feasible approach to achieving a bioactive and cost effective bioscavenger MCM.

Engineering death resistance in CHO cells for improved perfusion culture.

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

The potential of long noncoding RNA therapies.

The human genome expresses vast numbers of long noncoding RNAs (lncRNA) that fulfil diverse roles in gene regulation, cell biology, development, and human disease. These roles are often mediated by sequence motifs and secondary structures bound by proteins and can regulate epigenetic, transcriptional, and translational pathways. These functional domains can be further optimised and engineered into RNA devices that are widely used in synthetic biology. We propose that natural lncRNA structures can be explored and exploited for the rational design and assembly of synthetic RNA therapies. This potential has been enabled by advances in the stability, immunogenicity, manufacture, and delivery of other RNA-based therapies, from which we can anticipate the pharmacological properties of lncRNA therapies that have not yet otherwise entered clinical trials.

Modeling apoptosis resistance in CHO cells with CRISPR-mediated knockouts of Bak1, Bax, and Bok.

Chinese hamster ovary (CHO) cells are the primary platform for the production of biopharmaceuticals. To increase yields, many CHO cell lines have been genetically engineered to resist cell death. However, the kinetics that governs cell fate in bioreactors are confounded by many variables associated with batch processes. Here, we used CRISPR-Cas9 to create combinatorial knockouts of the three known BCL-2 family effector proteins: Bak1, Bax, and Bok. To assess the response to apoptotic stimuli, cell lines were cultured in the presence of four cytotoxic compounds with different mechanisms of action. A population-based model was developed to describe the behavior of the resulting viable cell dynamics as a function of genotype and treatment. Our results validated the synergistic antiapoptotic nature of Bak1 and Bax, while the deletion of Bok had no significant impact. Importantly, the uniform application of apoptotic stresses permitted direct observation and quantification of a delay in the onset of cell death through Bayesian inference of meaningful model parameters. In addition to the classical death rate, a delay function was found to be essential in the accurate modeling of the cell death response. These findings represent an important bridge between cell line engineering strategies and biological modeling in a bioprocess context.

Perfusion culture of Chinese Hamster Ovary cells for bioprocessing applications.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.

Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer.

Optimal cytoreduction for ovarian cancer is often challenging because of aggressive tumor biology and advanced stage. It is a critical issue since the extent of residual disease after surgery is the key predictor of ovarian cancer patient survival. For a limited number of cancers, fluorescence-guided surgery has emerged as an effective aid for tumor delineation and effective cytoreduction. The intravenously administered fluorescent agent, most commonly indocyanine green (ICG), accumulates preferentially in tumors, which are visualized under a fluorescent light source to aid surgery. Insufficient tumor specificity has limited the broad application of these agents in surgical oncology including for ovarian cancer. In this study, we developed a novel tumor-selective fluorescent agent by chemically linking ICG to mouse monoclonal antibody 10D7 that specifically recognizes an ovarian cancer-enriched cell surface receptor, CUB-domain-containing protein 1 (CDCP1). 10D7ICG has high affinity for purified recombinant CDCP1 and CDCP1 that is located on the surface of ovarian cancer cells in vitro and in vivo. Our results show that intravenously administered 10D7ICG accumulates preferentially in ovarian cancer, permitting visualization of xenograft tumors in mice. The data suggest CDCP1 as a rational target for tumor-specific fluorescence-guided surgery for ovarian cancer.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Safety and immunogenicity of an MF59-adjuvanted spike glycoprotein-clamp vaccine for SARS-CoV-2: a randomised, double-blind, placebo-controlled, phase 1 trial.

BACKGROUND: Given the scale of the ongoing COVID-19 pandemic, the development of vaccines based on different platforms is essential, particularly in light of emerging viral variants, the absence of information on vaccine-induced immune durability, and potential paediatric use. We aimed to assess the safety and immunogenicity of an MF59-adjuvanted subunit vaccine for COVID-19 based on recombinant SARS-CoV-2 spike glycoprotein stabilised in a pre-fusion conformation by a novel molecular clamp (spike glycoprotein-clamp [sclamp]). METHODS: We did a phase 1, double-blind, placebo-controlled, block-randomised trial of the sclamp subunit vaccine in a single clinical trial site in Brisbane, QLD, Australia. Healthy adults (aged ≥18 to ≤55 years) who had tested negative for SARS-CoV-2, reported no close contact with anyone with active or previous SARS-CoV-2 infection, and tested negative for pre-existing SARS-CoV-2 immunity were included. Participants were randomly assigned to one of five treatment groups and received two doses via intramuscular injection 28 days apart of either placebo, sclamp vaccine at 5 µg, 15 µg, or 45 µg, or one dose of sclamp vaccine at 45 µg followed by placebo. Participants and study personnel, except the dose administration personnel, were masked to treatment. The primary safety endpoints included solicited local and systemic adverse events in the 7 days after each dose and unsolicited adverse events up to 12 months after dosing. Here, data are reported up until day 57. Primary immunogenicity endpoints were antigen-specific IgG ELISA and SARS-CoV-2 microneutralisation assays assessed at 28 days after each dose. The study is ongoing and registered with ClinicalTrials.gov, NCT04495933. FINDINGS: Between June 23, 2020, and Aug 17, 2020, of 314 healthy volunteers screened, 120 were randomly assigned (n=24 per group), and 114 (95%) completed the study up to day 57 (mean age 32·5 years [SD 10·4], 65 [54%] male, 55 [46%] female). Severe solicited reactions were infrequent and occurred at similar rates in participants receiving placebo (two [8%] of 24) and the SARS-CoV-2 sclamp vaccine at any dose (three [3%] of 96). Both solicited reactions and unsolicited adverse events occurred at a similar frequency in participants receiving placebo and the SARS-CoV-2 sclamp vaccine. Solicited reactions occurred in 19 (79%) of 24 participants receiving placebo and 86 (90%) of 96 receiving the SARS-CoV-2 sclamp vaccine at any dose. Unsolicited adverse events occurred in seven (29%) of 24 participants receiving placebo and 35 (36%) of 96 participants receiving the SARS-CoV-2 sclamp vaccine at any dose. Vaccination with SARS-CoV-2 sclamp elicited a similar antigen-specific response irrespective of dose: 4 weeks after the initial dose (day 29) with 5 µg dose (geometric mean titre [GMT] 6400, 95% CI 3683-11 122), with 15 µg dose (7492, 4959-11 319), and the two 45 µg dose cohorts (8770, 5526-13 920 in the two-dose 45 µg cohort; 8793, 5570-13 881 in the single-dose 45 µg cohort); 4 weeks after the second dose (day 57) with two 5 µg doses (102 400, 64 857-161 676), with two 15 µg doses (74 725, 51 300-108 847), with two 45 µg doses (79 586, 55 430-114 268), only a single 45 µg dose (4795, 2858-8043). At day 57, 67 (99%) of 68 participants who received two doses of sclamp vaccine at any concentration produced a neutralising immune response, compared with six (25%) of 24 who received a single 45 µg dose and none of 22 who received placebo. Participants receiving two doses of sclamp vaccine elicited similar neutralisation titres, irrespective of dose: two 5 µg doses (GMT 228, 95% CI 146-356), two 15 µg doses (230, 170-312), and two 45 µg doses (239, 187-307). INTERPRETATION: This first-in-human trial shows that a subunit vaccine comprising mammalian cell culture-derived, MF59-adjuvanted, molecular clamp-stabilised recombinant spike protein elicits strong immune responses with a promising safety profile. However, the glycoprotein 41 peptide present in the clamp created HIV diagnostic assay interference, a possible barrier to widespread use highlighting the criticality of potential non-spike directed immunogenicity during vaccine development. Studies are ongoing with alternative molecular clamp trimerisation domains to ameliorate this response. FUNDING: Coalition for Epidemic Preparedness Innovations, National Health and Medical Research Council, Queensland Government, and further philanthropic sources listed in the acknowledgments.

Mutational and biophysical robustness in a prestabilized monobody.

The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Working Hard or Hardly Working? Regulatory Bottlenecks in Developing a COVID-19 Vaccine.

Vaccine solutions rarely reach the public until after an outbreak abates; an Ebola vaccine was approved 5 years after peak outbreak and SARS, MERS, and Zika vaccines are still in clinical development. Despite massive leaps forward in rapid science, other regulatory bottlenecks are hamstringing the global effort for pandemic vaccines.