Development Of Biotherapeutics Research Articles

Harnessing the potential of 2-methyl imidazolium dihydrogen phosphate as a protein stabilizer: Assessing its toxicity and impact on aggregation, structural integrity, and functional efficacy of monoclonal antibodies

The development of safe and effective biotherapeutics faces challenges due to protein aggregation. These aggregates, formed due to various stress factors, can lead to irreversible changes and increase the risk of immunogenicity and off-target binding. Ionic liquids (ILs) have recently gained significant attention due to their potential in stabilizing proteins and enzymes. In this investigation, we have assessed the thermal stability of bevacizumab (BmAb), in presence of 2-methyl imidazolium dihydrogen phosphate (2- MIDHP), both alone and in combination with other formulation excipients. SEC, DLS, FFF-MALS and AUC were used to study the protective effect of 2-MIDHP on BmAb aggregation. CD and fluorescence spectroscopy were used to evaluate the impact of 2-MIDHP on structural changes in BmAb, while SPR was used to assess the binding activity in the presence and absence of 2-MIDHP. Subsequently, a cell viability assay (MTT) was conducted to evaluate potential toxic effects of 2-MIDHP with BmAb. Overall, our findings suggested the potential of 2-MIDHP in enhancing the stability of BmAb without any negative impact on its structural and binding activity, and its compatibility with other formulation components, thus indicating its promising prospects for the development of improved biotherapeutics.

Systematic Optimization of Activity-Based Protein Profiling for Identification of Polysorbate-Degradative Enzymes in Biotherapeutic Drug Substance down to 10 ppb.

The identification and control of high-risk host cell proteins (HCPs) in biotherapeutics development are crucial for ensuring product quality and shelf life. Specifically, HCPs with hydrolase activity can cause the degradation of excipient polysorbates (PS), leading to a decrease in the shelf life of the drug product. In this study, we systematically optimized every step of an activity-based protein profiling (ABPP) workflow to identify trace amounts of active polysorbate-degradative enzymes (PSDEs) in biotherapeutic process intermediates. Evaluation of various parameters during sample preparation pinpointed the optimal pH level and fluorophosphonate (FP)-biotin concentration. Moreover, the combined use of a short liquid chromatography gradient and the fast-scanning parallel accumulation-serial fragmentation (PASEF) methodology increased sample throughput without compromising identification coverage. Tuning the trapped ion mobility spectrometry (TIMS) parameters further enhanced sensitivity. In addition, we evaluated various data acquisition modes, including PASEF combined with data-dependent acquisition (DDA PASEF), data-independent acquisition (diaPASEF), or parallel reaction monitoring (prm-PASEF). By employing the newly optimized ABPP workflow, we successfully identified PSDEs at a concentration as low as 10 ppb in a drug substance sample. Finally, the new workflow enabled us to detect a PSDE that could not be detected with the original workflow during a PS degradation root-cause investigation.

Evaluating the influence of the initial high molecular weight level on monoclonal antibody particle formation kinetics using a short-term chemical stress study

Protein formulations may form proteinaceous particles that vary in size from nanometers to millimeters. Monitoring the kinetics of protein particle formation, e.g., through accelerated degradation studies, is an attempt to understand and assess the rate and progression of particle populations. Little is known about whether the initial level of high molecular weight (HMW) species, or initial HMW level (IHL), of a protein solution influences the propagation of protein particle formation, and thus affects the storage stability of proteins.In this study, we have established a method to generate protein solutions of different IHLs by thermal stress. We have evaluated a 16-week thermal stability study at 40 °C of two monoclonal antibodies (mAb-A and mAb-B) at different IHLs using size exclusion chromatography (SEC) and sub-visible particle analysis. We have performed an isothermal stress study with guanidinium hydrochloride (GuaHCl) at room temperature for 300-min to evaluate the formation of HMWs analysed by SEC. The application of the Finke-Watzky (F-W) two-step nucleation model allowed us to mathematically describe the kinetics of HMW formation and to extract kinetic parameters of this process.For mAb-A, the IHLs had a marginal influence on the loss of monomer rate; instead, mAb-A exhibited fragmentation at 40 °C, which was independent of the IHL. Nevertheless, above a threshold of ≥ 7 % IHL, existing trimers/tetramers undergo conversion into higher-order oligomers at 40 °C, which is not observed at lower IHLs. In contrast, mAb-B exhibited an increased HMW formation rate above a threshold of ≥ 4 % IHL, which was reflected in the monomer decay rates at 40 °C and the F-W kinetic parameters of the chemical stress study.This case study shows that the initial level of HMWs exerts a differential influence on the progression of HMW formation. In one instance, there is a discernible acceleration in the formation of HMWs with rising IHLs. Conversely, in another example, the IHL exerts only a slight influence on HMW formation. Moreover, the results of our short-term chemical stress study are in accordance with those of a classical storage stability study conducted at 40 °C, which evaluated different IHLs. The analysis of HMW formation kinetics will enhance our understanding of the protein particle formation process and facilitate the formulation development of biotherapeutics.

An Integrated Strategy to Identify Tyrosine Sulfation from the Therapeutic Proteins.

Posttranslational modifications (PTMs) are potential critical quality attributes in biotherapeutic development, as they can affect drug efficacy and safety. Tyrosine sulfation plays a critical role in protein-protein interactions and has been found on many surface receptors as well as antibody complementarity-determining regions (CDR). However, the presence and function of tyrosine sulfation in therapeutic proteins have not been broadly investigated due to difficulties in detecting the modification. Here, we establish an integrated strategy to identify tyrosine sulfation in biotherapeutic proteins. In silico prediction was used to estimate possible modification sites, followed by the elucidation with intact LCMS and native SCX-MS. The combination of these three steps takes less than 1 h, which provides quick and confident preliminary detection of potential CQAs. Taking NB1 as an example, three +80 Da mass shifts were observed from intact mass analysis and three acidic peaks were monitored by SCX, allowing confirmation of modification as either phosphorylation or sulfation. Peptide mapping, Fe3+-IMAC enrichment, and dephosphorylation were further conducted to provide improved signal intensity and differentiation of modification such as sulfation or phosphorylation. With this integrated strategy, we were able to identify for the first time both tyrosine sulfation and serine phosphorylation in one therapeutic protein.

Advancements in mammalian display technology for therapeutic antibody development and beyond: current landscape, challenges, and future prospects.

The evolving development landscape of biotherapeutics and their growing complexity from simple antibodies into bi- and multi-specific molecules necessitates sophisticated discovery and engineering platforms. This review focuses on mammalian display technology as a potential solution to the pressing challenges in biotherapeutic development. We provide a comparative analysis with established methodologies, highlighting key aspects of mammalian display technology, including genetic engineering, construction of display libraries, and its pivotal role in hit selection and/or developability engineering. The review delves into the mechanisms underpinning developability-driven selection via mammalian display and their broader implications. Applications beyond antibody discovery are also explored, alongside advancements towards function-first screening technologies, precision genome engineering and AI/ML-enhanced libraries, situating them in the context of mammalian display. Overall, the review provides a comprehensive overview of the current mammalian display technology landscape, underscores the expansive potential of the technology for biotherapeutic development, addresses the critical challenges for the full realisation of this potential, and examines advances in related disciplines that might impact the future application of mammalian display technologies.

Double-layer dissolving microneedles for delivery of mesenchymal stem cell Secretome: Formulation, characterisation and skin irritation study

Regenerative therapy based on stem cells have been developed, focusing on either stem cell or secretome delivery. Most marketed cellular and gene therapy products are available as injectable dosage forms, leading to several limitations requiring alternative routes, such as the intradermal route. Microneedles, capable of penetratingthe stratum corneumbarrier, offer a potential alternative for intradermal delivery. This present study aimed to develop double-layer dissolving microneedles (DMN) for the delivery of freeze-dried mesenchymal stem cell secretome. DMNs were fabricated using a two-step casting method and composed of two polymer combinations: poly(vinyl pyrrolidone) (PVP) with poly(vinyl alcohol) (PVA) or PVP with sodium hyaluronate (SH). The manufactured DMNs underwent assessments for morphology, mechanical strength, in skin dissolution, protein content, in vitro permeation, in vivo skin irritation, and physical stability. Based on evaluations of morphology and mechanical strength, two formulas (F5 and F12) met acceptance criteria. Evaluation of protein content revealed that F12 (PVP-SH combination) had a higher protein content than F5 (PVP-PVA combination), 99.02 ± 3.24 μg and 78.36 ± 3.75 μg respectively. In vitro permeation studies showed that F5 delivered secretome protein by 100.84 ± 0.88%, while F12 delivered 99.63 ± 9.21% in 24 h. After four days of observation onSprague-Dawleyrat’s skin, no signs of irritation, such as oedema and redness, was observed after applying both formulations. The safety of using PVP-PVA and PVP-SH combinations as excipients for DMN secretome delivery has been confirmed, promising significant advancements in biotherapeutic development in the future.

Mouse embryo CoCoPUTs: novel murine transcriptomic-weighted usage website featuring multiple strains, tissues, and stages

Mouse (Mus musculus) models have been heavily utilized in developmental biology research to understand mammalian embryonic development, as mice share many genetic, physiological, and developmental characteristics with humans. New explorations into the integration of temporal (stage-specific) and transcriptional (tissue-specific) data have expanded our knowledge of mouse embryo tissue-specific gene functions. To better understand the substantial impact of synonymous mutational variations in the cell-state-specific transcriptome on a tissue’s codon and codon pair usage landscape, we have established a novel resource—Mouse Embryo Codon and Codon Pair Usage Tables (Mouse Embryo CoCoPUTs). This webpage not only offers codon and codon pair usage, but also GC, dinucleotide, and junction dinucleotide usage, encompassing four strains, 15 murine embryonic tissue groups, 18 Theiler stages, and 26 embryonic days. Here, we leverage Mouse Embryo CoCoPUTs and employ the use of heatmaps to depict usage changes over time and a comparison to human usage for each strain and embryonic time point, highlighting unique differences and similarities. The usage similarities found between mouse and human central nervous system data highlight the translation for projects leveraging mouse models. Data for this analysis can be directly retrieved from Mouse Embryo CoCoPUTs. This cutting-edge resource plays a crucial role in deciphering the complex interplay between usage patterns and embryonic development, offering valuable insights into variation across diverse tissues, strains, and stages. Its applications extend across multiple domains, with notable advantages for biotherapeutic development, where optimizing codon usage can enhance protein expression; one can compare strains, tissues, and mouse embryonic stages in one query. Additionally, Mouse Embryo CoCoPUTs holds great potential in the field of tissue-specific genetic engineering, providing insights for tailoring gene expression to specific tissues for targeted interventions. Furthermore, this resource may enhance our understanding of the nuanced connections between usage biases and tissue-specific gene function, contributing to the development of more accurate predictive models for genetic disorders.

Internalization of therapeutic antibodies into dendritic cells as a risk factor for immunogenicity.

Immunogenicity, the unwanted immune response triggered by therapeutic antibodies, poses significant challenges in biotherapeutic development. This response can lead to the production of anti-drug antibodies, potentially compromising the efficacy and safety of treatments. The internalization of therapeutic antibodies into dendritic cells (DCs) is a critical factor influencing immunogenicity. Using monoclonal antibodies, with differences in non-specific cellular uptake, as tools to explore the impact on the overall risk of immunogenicity, this study explores how internalization influences peptide presentation and subsequently T cell activation. To investigate the impact of antibody internalization on immunogenicity, untargeted toolantibodies with engineered positive or negative charge patches were utilized. Immature monocyte-derived DCs (moDCs), known for their physiologically relevant high endocytic activity, were employed for internalization assays, while mature moDCs were used for MHC-II associated peptide proteomics (MAPPs) assays. In addition to the lysosomal accumulation and peptide presentation, subsequent CD4+ T cell activation has been assessed. Consequently, a known CD4+ T cell epitope from ovalbumin was inserted into the tool antibodies to evaluate T cell activation on a single, shared epitope. Antibodies with positive charge patches exhibited higher rates of lysosomal accumulation and epitope presentation compared to those with negative charge patches or neutral surface charge. Furthermore, a direct correlation between internalization rate and presentation on MHC-II molecules could be established. To explore the link between internalization, peptide presentation and CD4+ T cell activation, tool antibodies containing the same OVA epitope were used. Previous observations were not altered by the insertion of the OVA epitope and ultimately, an enhanced CD4+ T cell response correlated with increased internalization in DCs and peptide presentation. These findings demonstrate that the biophysical properties of therapeutic antibodies, particularly surface charge, play a crucial role in their internalization into DCs. Antibodies internalized faster and processed by DCs, are also more prone to be presented on their surface leading to a higher risk of triggering an immune response. These insights underscore the importance of considering antibody surface charge and other properties that enhance cellular accumulation during the preclinical development of biotherapeutics to mitigate immunogenicity risks.

Concept for recycling a small-scale plastic-based bioreactor in a close-loop – Technical approach

To accelerate biotherapeutic development whilst decreasing development time and project cost, small-scale multi-parallel bioreactors have been developed to maximize product yield and quality. These systems feature single-use plastic-based bioreactors for easy connection and fast turnaround. Despite well-known advantages, the perception of plastic and the lack of recycling options are raising concerns in the scientific laboratories’ community. One of the common objections to plastic circularity in life-science is the perceived “downcycling” effect of mechanical recycling. Therefore, the aim of this work was to establish the technical feasibility of a close-loop recycling concept for the main construction material of the small-scale bioreactor. Application tests (cell compatibility and cell culture) were performed and supported by quantitative physical and mechanical tests (tensile, melt flow index, light transmission). Results show that mechanically recycled polycarbonate could be re-used in the same application. A comparative life cycle assessment (LCA), based on a theoretical framework, showed with different scenarios that recycling would have a positive impact on Climate – Carbon – total within the boundaries. Even if the technical feasibility of such a concept is demonstrated through this study, several challenges remain for such a closed-loop recycling concept to be implemented at a commercial scale.

Abstract Mo014: Cardioactive molecules identified by functional in vivo screening prevent anthracycline cardiotoxicity

Introduction: Promotion of cardiomyocyte survival upon insult is mandatory to allow maintenance of ventricular function and dam development of severe heart dysfunction. Recently, through an in vivo functional screening of the mouse secretome we identified potent mediators of cardiomyocyte survival among 1200 soluble factors expressed by AAV vectors. The cardiac overexpression of the most effective cytokines (Chrdl1, Fam3c and Fam3b) maintained cardiac function after myocardial infarction by blunting oxidative damage, decreasing cell death, promoting autophagy, and inhibiting myofibroblast activation (Ruozi, Bortolotti et al. 2022. Sci Transl Med 14, eabo0699) Aim: We investigated the effect of cardioactive proteins in a mouse anthracycline model of cardiotoxicity. Material and Methods: Mimicking clinical chronic therapeutic regimens, doxorubicin (24 mg/kg) was administered to C57/BL6 female mice one week after AAV9-driven cardiac overexpression of Chrdl1, Fam3b or Fam3c. Echocardiography was performed after 6 and 8 weeks and was followed by histology and molecular analysis. Apoptosis, autophagy and mitochondrial function was analysed in primary cardiomyocytes along with pathway investigation. Results: AAV9-mediated overexpression of Chrdl1, Fam3b and Fam3c markedly improved animal survival. Echocardiographic assessment revealed significant reduction of left ventricular ejection fraction and marked heart dilatation in control animals, while all treated animals maintained functional and anatomical parameters. Treated mice showed normal cardiac mass and cardiomyocyte cross-sectional area, reduced interstitial collagen deposition and decreased expression of profibrotic genes. In both rodents and human cardiomyocytes, Chrdl1, Fam3b and Fam3c factors decreased doxorubicin induced apoptosis, induced protective macroautophagy and preserved mitochondrial morphology. Chrdl1 prevented doxorubicin-mediated BMP upregulation and autophagy engulfment through the inhibition of a detrimental molecular pathway downstream the BMP receptor. Conclusions: The development of biotherapeutics that act as pro-survival factors for cardiomyocytes offers significant translational perspectives in cardio-oncology.

Pulmonary Inhalation of Biotherapeutics: A Systematic Approach to Understanding the Effects of Atomisation Gas Flow Rate on Particle Physiochemical Properties and Retained Bioactivity.

The identification of spray-drying processing parameters capable of producing particles suitable for pulmonary inhalation with retained bioactivity underpins the development of inhalable biotherapeutics. Effective delivery of biopharmaceuticals via pulmonary delivery routes such as dry powder inhalation (DPI) requires developing techniques that engineer particles to well-defined target profiles while simultaneously minimising protein denaturation. This study examines the simultaneous effects of atomisation gas flow rate on particle properties and retained bioactivity for the model biopharmaceutical lysozyme. The results show that optimising the interplay between atomisation gas flow rate and excipient concentration enables the production of free-flowing powder with retained bioactivity approaching 100%, moisture content below 4%, and D50 < 4 µm, at yields exceeding 50%. The developed methodologies inform the future design of protein-specific spray-drying parameters for inhalable biotherapeutics.

Indigenous Medicinal Plants Based Remedies in Management of Arthritis by &lt;i&gt;Irula&lt;/i&gt; Tribals of Tamil Nadu – A Comprehensive Review

Arthritis is a chronic autoimmune illness that causes joint dysfunction, and antibodies directed against self-neoepitopes. Globally more than 350 million peoples have arthritis and it is the second most rheumatological problem with a prevalence of 22% to 39% in India reported by World Health Organization. Considering the numerous synthetic therapeutic strategies that target immune cells and cytokines but drug administration of allopathic anti-arthritic medicaments is still difficult because of the persistent hazardous consequences. Presently, the use of medicinal plants as a trustworthy source of new therapy has become more popular due to the numerous side effects of synthetic drugs. One of the most significant foes in modern medicine, non steroidal anti-inflammatory drugs creates huge impact on human health, high influence on living quality and has significant economic implications. National Biopharma Mission primarily focus on the development of Bio-Therapeutics to achieve the sustainable health. According to the estimations based on various literatures, Fabaceae is the most significant herbal family which includes a vast range of plants having biological and therapeutic uses. This study presents the extensive knowledge about arthritis, immunomodulatory mechanism and existing drugs and therapies. The analgesic activity, potential active chemicals from the Fabaceae genus, as well as potential mechanisms of action for these plants are studied. The scientific information and evidences presented in this review paper is crucial for determining the precise effects of Fabaceae plants on pain modulation especially for arthritic pain and anti-inflammatory properties. This could be used to separate potentially active compounds from some of these medicinal plants in the future and create synthetic therapeutics.

Monoclonal Antibody Generation Using Single BCell Screening for Treating Infectious Diseases.

The screening of antigen-specific B cells has been pivotal for biotherapeutic development for over four decades. Conventional antibody discovery strategies, including hybridoma technology and single B cell screening, remain widely used based on their simplicity, accessibility, and proven track record. Technological advances and the urgent demand for infectious disease applications have shifted paradigms in single B cell screening, resulting in increased throughput and decreased time and labor, ultimately enabling the rapid identification of monoclonal antibodies with desired biological and biophysical properties. Herein, we provide an overview of conventional and emergent single B cell screening approaches and highlight their potential strengths and weaknesses. We also detail the impact of innovative technologies-including miniaturization, microfluidics, multiplexing, and deep sequencing-on the recent identification of broadly neutralizing antibodies for infectious disease applications. Overall, the coronavirus disease 2019 (COVID-19) pandemic has reinvigorated efforts to improve the efficiency of monoclonal antibody discovery, resulting in the broad application of innovative antibody discovery methodologies for treating a myriad of infectious diseases and pathological conditions.

Comparing Cell Penetration of Biotherapeutics across Human Cell Lines.

A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure that data could be compared across cell lines. We compared the nuclear penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

Antimicrobial therapeutic protein extraction from fruit waste and recent trends in their utilization against infections.

Fruits are a very good source of various nutrients that can boost overall human health. In these days, the recovery of therapeutic compounds from different fruit wastes is trending in research, which might not only minimize the waste problem but also encounter a higher demand for various enzymes that could have antimicrobial properties against infectious diseases. The goal of this review is to focus on the recovery of therapeutic enzymes from fruit wastes and its present-day tendency for utilization. Here we discussed different parts of fruit waste, such as pulp, pomace, seed, kernel, peel, etc., that produce therapeutic enzymes like amylase, cellulose, lipase, laccase, pectinase, etc. These bioactive enzymes are present in different parts of fruit and could be used as therapeutics against various infectious diseases. This article provides a thorough knowledge compilation of therapeutic enzyme isolation from fruit waste on a single platform, distinctly informative, and significant review work on the topic that is envisioned to encourage further research ideas in these areas that are still under-explored. This paper explains the various aspects of enzyme isolation from fruit and vegetable waste and their biotherapeutic potential that could provide new insights into the development of biotherapeutics and attract the attention of researchers to enhance translational research magnitude further.

Characterization of Therapeutic Antibody Charge Heterogeneity Under Stress Conditions by Microfluidic Capillary Electrophoresis Coupled with Mass Spectrometry

Therapeutic antibodies are a major class of biopharmaceutics that are applied in disease treatment because of their many advantages, including high specificity and high affinity to molecular targets. Between their production and administration, therapeutic antibodies are exposed to multiple stress conditions. Forced degradation and stress stability studies are conducted to simulate the risk of degradation and the effects of these stresses, thereby enhancing understanding of the drug product to support strategies to mitigate the impact from stressed conditions. These types of studies are also routinely conducted to evaluate product comparability when major process changes are implemented during the production. Charge variant analysis helps understand the changes in the electrostatic environment of biotherapeutics and can uncover underlying molecular level alterations associated with charge variants. Herein, we used ZipChip native capillary electrophoresis-mass spectrometry (nCE-MS) to elucidate the changes in charge variant profiles at the molecular level. In two case studies under thermal stress conditions, we observed that charge variants arose from both post-translational modifications (including deamidation, oxidation, and pyroglutamate formation) and sequence truncations at the hinge regions. Under oxidative stress conditions, oxidation was found to be the major contributor to the changes in the charge variant profiles. Under pH stress conditions, the changes in the charge variant profile were due to increased levels of deamidation, oxidation, and pyroglutamate formation. ZipChip nCE-MS analysis enables identification of charge variant species under various stress conditions, thus supporting process and formulation development of biotherapeutics.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

Characterization and immunogenicity assessment of MERS-CoV pre-fusion spike trimeric oligomers as vaccine immunogen

ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal beta-coronavirus that emerged in 2012. The virus is part of the WHO blueprint priority list with a concerning fatality rate of 35%. Scientific efforts are ongoing for the development of vaccines, anti-viral and biotherapeutics, which are majorly directed toward the structural spike protein. However, the ongoing effort is challenging due to conformational instability of the spike protein and the evasion strategy posed by the MERS-CoV. In this study, we have expressed and purified the MERS-CoV pre-fusion spike protein in the Expi293F mammalian expression system. The purified protein was extensively characterized for its biochemical and biophysical properties. Thermal stability analysis showed a melting temperature of 58°C and the protein resisted major structural changes at elevated temperature as revealed by fluorescence spectroscopy and circular dichroism. Immunological assessment of the MERS-CoV spike immunogen in BALB/c mice with AddaVaxTM and Imject alum adjuvants showed elicitation of high titer antibody responses but a more balanced Th1/Th2 response with AddaVaxTM squalene like adjuvant. Together, our results suggest the formation of higher-order trimeric pre-fusion MERS-CoV spike proteins, which were able to induce robust immune responses. The comprehensive characterization of MERS-CoV spike protein warrants a better understanding of MERS spike protein and future vaccine development efforts.

Carbene Footprinting Directs Design of Genetically Encoded Proximity-Reactive Protein Binders.

Genetically encoding proximal-reactive unnatural amino acids (PrUaas), such as fluorosulfate-l-tyrosine (FSY), into natural proteins of interest (POI) confer the POI with the ability to covalently bind to its interacting proteins (IPs). The PrUaa-incorporated POIs hold promise for blocking undesirable POI-IP interactions. Selecting appropriate PrUaa anchor sites is crucial, but it remains challenging with the current methodology, which heavily relies on crystallography to identify the proximal residues between the POIs and the IPs for the PrUaa anchorage. To address the challenge, here, we propose a footprinting-directed genetically encoded covalent binder (footprinting-GECB) approach. This approach employs carbene footprinting, a structural mass spectrometry (MS) technique that quantifies the extent of labeling of the POI following the addition of its IP, and thus identifies the responsive residues. By genetically encoding PrUaa into these responsive sites, POI variants with covalent bonding ability to its IP can be produced without the need for crystallography. Using the POI-IP model, KRAS/RAF1, we showed that engineering FSY at the footprint-assigned KRAS residue resulted in a KRAS variant that can bind irreversibly to RAF1. Additionally, we inserted FSY at the responsive residue in RAF1 upon footprinting the oncogenic KRASG12D/RAF1, which lacks crystal structure, and generated a covalent binder to KRASG12D. Together, we demonstrated that by adopting carbene footprinting to direct PrUaa anchorage, we can greatly expand the opportunities for designing covalent protein binders for PPIs without relying on crystallography. This holds promise for creating effective PPI inhibitors and supports both fundamental research and biotherapeutics development.

Engineering Mycoplasma pneumoniae to bypass the association with Guillain-Barré syndrome

A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.