30 Years of Biotherapeutics Development-What Have We Learned?

Since the birth of biotechnology, hundreds of biotherapeutics have been developed and approved by the US Food and Drug Administration (FDA) for human use. These novel medicines not only bring significant benefit to patients but also represent precision tools to interrogate human disease biology. Accordingly, much has been learned from the successes and failures of hundreds of high-quality clinical trials. In this review, we discuss general and broadly applicable themes that have emerged from this collective experience. We base our discussion on insights gained from exploring some of the most important target classes, including interleukin-1 (IL-1), tumor necrosis factor α (TNF-α), IL-6, IL-12/23, IL-17, IL-4/13, IL-5, immunoglobulin E (IgE), integrins and B cells. We also describe current challenges and speculate about how emerging technological capabilities may enable the discovery and development of the next generation of biotherapeutics.

Leveraging Cross-Linking Mass Spectrometry for Modeling Antibody-Antigen Complexes.

Elucidating antibody-antigen complexes at the atomic level is of utmost interest for understanding immune responses and designing better therapies. Cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for mapping protein-protein interactions, suggesting valuable structural insights. However, the use of XL-MS studies to enable epitope/paratope mapping of antibody-antigen complexes is still limited up to now. XL-MS data can be used to drive integrative modeling of antibody-antigen complexes, where cross-links information serves as distance restraints for the precise determination of binding interfaces. In this approach, XL-MS data are employed to identify connections between binding interfaces of the antibody and the antigen, thus informing molecular modeling. Current literature provides minimal input about the impact of XL-MS data on the integrative modeling of antibody-antigen complexes. Here, we applied XL-MS to retrieve information about binding interfaces of three antibody-antigen complexes. We leveraged XL-MS data to perform integrative modeling using HADDOCK (active-passive residues and distance restraints strategies) and AlphaLink2. We then compared these three approaches with initial predictions of investigated antibody-antigen complexes by AlphaFold Multimer. This work emphasizes the importance of cross-linking data in resolving conformational dynamics of antibody-antigen complexes, ultimately enhancing the design of better protein therapeutics and vaccines.

The NMR signature of maltose-based glycation in full-length proteins.

Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d4 in D2O. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods.

Modulating the Microbiome for Crohn's Disease Treatment.

The central role of the gut microbiota in the regulation of health and disease has been convincingly demonstrated. Polymicrobial interkingdom interactions between bacterial (the bacteriome) and fungal (the mycobiome) communities of the gut have become a prominent focus for development of potential therapeutic approaches. In addition to polymicrobial interactions, the complex gut ecosystem also mediates interactions between the host and the microbiota. These interactions are complex and bidirectional; microbiota composition can be influenced by host immune response, disease-specific therapeutics, antimicrobial drugs, and overall ecosystems. However, the gut microbiota also influences host immune response to a drug or therapy by potentially transforming the drug's structure and altering bioavailability, activity, or toxicity. This is especially true in cases where the gut microbiota has produced a biofilm. The negative ramifications of biofilm formation include alteration of gut permeability, enhanced antimicrobial resistance, and alteration of host immune response effectiveness. Natural modulation of the gut microbiota, using probiotic and prebiotic approaches, may also be used to affect the host microbiome, a type of "natural" modulation of the host microbiota composition. In this review, we discuss potential bidirectional interactions between microbes and host, and we describe the changes in gut microbiota induced by probiotic and prebiotic approaches as well as their potential clinical consequences, including biofilm formation. We outline a systematic approach to designing probiotics capable of altering the host microbiota in disease states, using Crohn's disease as a model chronic disease. Understanding how the effective changes in the microbiome may enhance treatment efficacy may unlock the possibility of modulating the gut microbiome to improve treatment using a natural approach.

Liquid-phase separations coupled with ion mobility-mass spectrometry for next-generation biopharmaceutical analysis.

INTRODUCTION: The pharmaceutical industry continues to expand its search for innovative biotherapeutics. The comprehensive characterization of such therapeutics requires many analytical techniques to fully evaluate critical quality attributes, making analysis a bottleneck in discovery and development timelines. While thorough characterization is crucial for ensuring the safety and efficacy of biotherapeutics, there is a need to further streamline analytical characterization and expedite the overall timeline from discovery to market. AREAS COVERED: This review focuses on recent developments in liquid-phase separations coupled with ion mobility-mass spectrometry (IM-MS) for the development and characterization of biotherapeutics. We cover uses of IM-MS to improve the characterization of monoclonal antibodies, antibody-drug conjugates, host cell proteins, glycans, and nucleic acids. This discussion is based on an extensive literature search using Web of Science, Google Scholar, and SciFinder. EXPERT OPINION: IM-MS has the potential to enhance the depth and efficiency of biotherapeutic characterization by providing additional insights into conformational changes, post-translational modifications, and impurity profiles. The rapid timescale of IM-MS positions it well to enhance the information content of existing assays through its facile integration with standard liquid-phase separation techniques that are commonly used for biopharmaceutical analysis.

Bacterial expression of a designed single-chain IL-10 prevents severe lung inflammation.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is active as a swapped domain dimer and is used in bacterial therapy of gut inflammation. IL-10 can be used as treatment of a wide range of pulmonary diseases. Here we have developed a non-pathogenic chassis (CV8) of the human lung bacterium Mycoplasma pneumoniae (MPN) to treat lung diseases. We find that IL-10 expression by MPN has a limited impact on the lung inflammatory response in mice. To solve these issues, we rationally designed a single-chain IL-10 (SC-IL10) with or without surface mutations, using our protein design software (ModelX and FoldX). As compared to the IL-10 WT, the designed SC-IL10 molecules increase the effective expression in MPN four-fold, and the activity in mouse and human cell lines between 10 and 60 times, depending on the cell line. The SC-IL10 molecules expressed in the mouse lung by CV8 in vivo have a powerful anti-inflammatory effect on Pseudomonas aeruginosa lung infection. This rational design strategy could be used to other molecules with immunomodulatory properties used in bacterial therapy.

Translation of nonclinical to clinical safety findings for 27 biotherapeutics.

Human adverse drug reactions (ADRs), and in vivo nonclinical adverse and nonadverse findings, were identified in 27 biotherapeutic programs and placed into organ categories to determine translation. The sensitivity of detecting human ADRs was 30.8% with a positive predictive value (PPV) of 53.3% for nonclinical adverse findings; sensitivity increased to 67.3% and PPV fell to 35.0% when including nonadverse findings. Nonclinical findings were associated with a greater likelihood of a human ADR in that organ category, especially for adverse findings [positive likelihood ratio (LR+) >10 (lower 95% confidence interval [CI] of >5)]. The specificity and negative predictive value (NPV) were very high (>85%). A lack of nonclinical findings in an organ category was associated with a lower likelihood of a human ADR in that organ category. About 40-50% of human ADRs and nonclinical adverse findings, and about 30% of nonclinical nonadverse findings, were attributed to pharmacology. Slightly more than half of the human ADRs with a translating nonclinical finding had findings in animals that could be considered very similar. Overall, 38% of nonclinical findings translated to a human ADR at the organ category level. When nonclinical findings did not translate to humans, the cause was usually higher exposures or longer dosing in animals. All programs with human ADRs attributed to immunogenicity also had nonclinical adverse or nonadverse findings related to immunogenicity. Overall, nonclinical adverse and nonadverse findings were useful in predicting human ADRs, especially at an organ category level, and the majority of human ADRs were predicted by nonclinical toxicity studies.

Designer probiotics: Opening the new horizon in diagnosis and prevention of human diseases.

Probiotic microorganisms have been used for therapeutic purposes for over a century, and recent advances in biotechnology and genetic engineering have opened up new possibilities for developing therapeutic approaches using indigenous probiotic microorganisms. Diseases are often related to metabolic and immunological factors, which play a critical role in their onset. With the help of advanced genetic tools, probiotics can be modified to produce or secrete important therapeutic peptides directly into mucosal sites, increasing their effectiveness. One potential approach to enhancing human health is through the use of designer probiotics, which possess immunogenic characteristics. These genetically engineered probiotics hold promise in providing novel therapeutic options. In addition to their immunogenic properties, designer probiotics can also be equipped with sensors and genetic circuits, enabling them to detect a range of diseases with remarkable precision. Such capabilities may significantly advance disease diagnosis and management. Furthermore, designer probiotics have the potential to be used in diagnostic applications, offering a less invasive and more cost-effective alternative to conventional diagnostic techniques. This review offers an overview of the different functional aspects of the designer probiotics and their effectiveness on different diseases and also, we have emphasized their limitations and future implications. A comprehensive understanding of these functional attributes may pave the way for new avenues of prevention and the development of effective therapies for a range of diseases.

Predictive modeling for cell culture in commercial manufacturing of biotherapeutics.

The biopharmaceutical industry continually seeks advancements in the commercial manufacturing of therapeutic proteins, where mammalian cell culture plays a pivotal role. The current work presents a novel data-driven predictive modeling application designed to enhance the efficiency and predictability of cell culture processes in biotherapeutic production. The capability of the cloud-based digital data science application, developed using open-source tools, is demonstrated with respect to predicting bioreactor potency from at-line process parameters over a 5-day horizon. The uncertainty in model's prediction is quantified, providing valuable insights for process control and decision-making. Model validation on unseen data confirms the model's robust generalizability. An interactive dashboard, tailored to process scientist's requirements is also developed to streamline biopharmaceutical manufacturing processes, ultimately leading to enhanced productivity and product quality.

Microneedle and Polymeric Films: Delivery of Proteins, Peptides and Nucleic Acids.

In the last 20 years, protein, peptide and nucleic acid-based therapies have become the fastest growing sector in the pharmaceutical industry and play a vital role in disease therapy. However, the intrinsic sensitivity and large molecular sizes of biotherapeutics limit the available routes of administration. Currently, the main administration routes of biomacromolecules, such as parenteral, oral, pulmonary, nasal, rectal and buccal routes, each have their limitations. Several non-invasive strategies have been proposed to overcome these challenges. Researchers were particularly interested in microneedles (MNs) and polymeric films because of their less invasiveness, convenience and greater potential to preserve the bioactivity of biotherapeutics. By facilitating with MNs and polymeric films, biomacromolecules could provide significant benefits to patients suffering from various diseases such as cancer, diabetes, infectious and ocular diseases. However, before these devices can be used on patients, how to upscale MN manufacture in a cost-effective and timely manner, as well as the long-term safety of MN and polymeric film applications necessitates further investigation.

Topical Prescription Management.

With recent advances in topical therapies for atopic dermatitis (AD), steroid-sparing options like calcineurin inhibitors, Janus kinase (JAK) inhibitors, and phosphodiesterase-4 (PDE-4) inhibitors are becoming mainstays in therapy, underscoring the importance of careful selection and usage of topical corticosteroids (TCSs) to minimize side effects. Alongside the necessity of emollient use, these steroid-sparing alternatives offer rapid itch relief and efficacy in improving disease severity. While TCSs still hold a prominent role in AD management, promising novel topical treatments like tapinarof and live biotherapeutics to modulate the skin microbiome are also discussed. Overall, the recent addition of novel topical therapies offers diverse options for AD management and underscores the importance of topical treatments in the management of AD.

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.

Function and regulation of cis P-tau in the pathogenesis and treatment of conventional and nonconventional tauopathies.

Conventional tauopathies are a group of disease characterized by tau inclusions in the brains, including Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and certain types of frontotemporal dementia (FTD), among which AD is the most prevalent. Extensive post-translational modifications, especially hyperphosphorylation, and abnormal aggregation of tau protein underlie tauopathy. Cis-trans isomerization of protein plays an important role in protein folding, function, and degradation, which is regulated by peptidyl-proline isomerases (PPIases). Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), the only PPIase found to isomerize Pro following phosphorylated Ser or Thr residues, alters phosphorylated tau protein conformation at pT231-P motif. The cis P-tau but not trans P-tau serves as an early driver of multiple neurodegenerative disease, encompassing AD, traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), and vascular contributions to cognitive impairment and dementia (VCID). Cis but not trans P-tau is resistant to protein dephosphorylation and degradation, and also prone to protein aggregation. Cis P-tau loses its ability to stabilize microtubule, causing and spreading tauopathy mainly in axons, a pathological process called cistauosis. The conformation-specific monoclonal antibody that targets only the cis P-tau serves as a very early diagnosis method and a potential treatment of not only conventional tauopathies but also nonconventional tauopathies such as VCID, with clinical trials ongoing. Notably, cis P-tau antibody is the only clinical-stage Alzheimer's therapeutic that has shown the efficacy in animal models of not only AD but also TBI and stroke, which are very early stages of dementia. Here we review the identification and pathological consequences of cis pt231-tau, the role of its regulator Pin1, as well as the clinical implication of cis pt231-tau conformation-specific antibody in conventional and nonconventional tauopathies.

Gene therapy in cancer.

Gene therapy, recently frequently investigated, is an alternative treatment method that introduces therapeutic genes into a cancer cell or tissue to cause cell death or slow down the growth of the cancer. This treatment has various strategies such as therapeutic gene activation or silencing of unwanted or defective genes; therefore a wide variety of genes and viral or nonviral vectors are being used in studies. Gene therapy strategies in cancer can be classified as inhibition of oncogene activation, activation of tumor suppressor gene, immunotherapy, suicide gene therapy and antiangiogenic gene therapy. In this review, we explain gene therapy, gene therapy strategies in cancer, approved gene medicines for cancer treatment and future of gene therapy in cancer. Today gene therapy has not yet reached the level of replacing conventional therapies. However, with a better understanding of the mechanism of cancer to determine the right treatment and target, in the future gene therapy, used as monotherapy or in combination with another existing treatment options, is likely to be used as a new medical procedure that will make cancer a controllable disease.

Engineered probiotics as live biotherapeutics for diagnosis and treatment of human diseases.

The use of probiotics to regulate the intestinal microbiota to prevent and treat a large number of disorders and diseases has been an international research hotspot. Although conventional probiotics have a certain regulatory role in nutrient metabolism, inhibiting pathogens, inducing immune regulation, and maintaining intestinal epithelial barrier function, they are unable to treat certain diseases. In recent years, aided by the continuous development of synthetic biology, engineering probiotics with desired characteristics and functionalities to benefit human health has made significant progress. In this article, we summarise the mechanism of action of conventional probiotics and their limitations and highlight the latest developments in the design and construction of probiotics as living diagnostics and therapeutics for the detection and treatment of a series of diseases, including pathogen infections, cancer, intestinal inflammation, metabolic disorders, vaccine delivery, cognitive health, and fatty liver. Besides we discuss the concerns regarding engineered probiotics and corresponding countermeasures and outline the desired features in the future development of engineered live biotherapeutics.

Embedding Dynamics in Intrinsic Physicochemical Profiles of Market-Stage Antibody-Based Biotherapeutics.

Adequate stability, manufacturability, and safety are crucial to bringing an antibody-based biotherapeutic to the market. Following the concept of holistic in silico developability, we introduce a physicochemical description of 91 market-stage antibody-based biotherapeutics based on orthogonal molecular properties of variable regions (Fvs) embedded in different simulation environments, mimicking conditions experienced by antibodies during manufacturing, formulation, and in vivo. In this work, the evaluation of molecular properties includes conformational flexibility of the Fvs using molecular dynamics (MD) simulations. The comparison between static homology models and simulations shows that MD significantly affects certain molecular descriptors like surface molecular patches. Moreover, the structural stability of a subset of Fv regions is linked to changes in their specific molecular interactions with ions in different experimental conditions. This is supported by the observation of differences in protein melting temperatures upon addition of NaCl. A DEvelopability Navigator In Silico (DENIS) is proposed to compare mAb candidates for their similarity with market-stage biotherapeutics in terms of physicochemical properties and conformational stability. Expanding on our previous developability guidelines (Ahmed et al. Proc. Natl. Acad. Sci. 2021, 118 (37), e2020577118), the hydrodynamic radius and the protein strand ratio are introduced as two additional descriptors that enable a more comprehensive in silico characterization of biotherapeutic drug candidates. Test cases show how this approach can facilitate identification and optimization of intrinsically developable lead candidates. DENIS represents an advanced computational tool to progress biotherapeutic drug candidates from discovery into early development by predicting drug properties in different aqueous environments.

Plant-derived nanoparticles and plant virus nanoparticles: Bioactivity, health management, and delivery potential.

Natural plants have acquired an increasing attention in biomedical research. Recent studies have revealed that plant-derived nanoparticles (PDNPs), which are nano-sized membrane vesicles released by plants, are one of the important material bases for the health promotion of natural plants. A great deal of research in this field has focused on nanoparticles derived from fresh vegetables and fruits. Generally, PDNPs contain lipids, proteins, nucleic acids, and other active small molecules and exhibit unique biological regulatory activity and editability. Specifically, they have emerged as important mediators of intercellular communication, and thus, are potentially suitable for therapeutic purposes. In this review, PDNPs were extensively explored; by evaluating them systematically starting from the origin and isolation, toward their characteristics, including morphological compositions, biological functions, and delivery potentials, as well as distinguishing them from plant-derived exosomes and highlighting the limitations of the current research. Meanwhile, we elucidated the variations in PDNPs infected by pathogenic microorganisms and emphasized on the biological functions and characteristics of plant virus nanoparticles. After clarifying these problems, it is beneficial to further research on PDNPs in the future and develop their clinical application value.

A Novel Method to Optimize Drug Delivery for Parenteral Products Involving New Therapies and Unmet Needs.

Healthcare trends continue to move from hospital to home and many targeted therapies and precision medicines are now being designed to be self-administered by patients or delivered in a home setting. In the case of long acting injectables and bio-therapeutics, the importance of optimal drug/biologic-device combination in terms of user needs becomes critical for successful clinical outcomes. The risk especially increases for novel therapies due to unknowns and uncertainties surrounding new formulation flow behavior, delivery methods, new injection sites and therapeutic optimization. Other risk factors include patient tolerability and acceptability. The success of the clinical outcome is now dependent on the optimal delivery in these situations for a consistent pharmacokinetics (PK) response. In addition, the complexity of formulations and challenging delivery needs have brought to light some of the limitations of existing legacy device technology, which may not be suited for these novel applications. The formulation itself may not be an exact fit to deliver with existing standard device technologies and may need to be designed to deliver the specific formulation appropriately. Formulations may have to be optimized both for delivery and for the therapeutic outcome creating many iterative development cycles. Rapid development of the therapies requires simultaneous development of drug and device and thus the importance of early-stage characterization. We present a novel integrated approach that includes drug delivery optimization via an autoinjector simulator in pre-clinical and clinical studies to assess the PK performance and potential to establish path to device early on and reduce time to the clinic.

Outlook on next-generation probiotics from the human gut.

Probiotics currently available on the market generally belong to a narrow range of microbial species. However, recent studies about the importance of the gut microbial commensals on human health highlighted that the gut microbiome is an unexplored reservoir of potentially beneficial microbes. For this reason, academic and industrial research is focused on identifying and testing novel microbial strains of gut origin for the development of next-generation probiotics. Although several of these are promising for the prevention and treatment of many chronic diseases, studies on human subjects are still scarce and approval from regulatory agencies is, therefore, rare. In addition, some issues need to be overcome before implementing their wide application on the market, such as the best methods for cultivation and storage of these oxygen-sensitive taxa. This review summarizes the most recent evidence related to NGPs and provides an outlook to the main issues that still limit their wide employment.

A highly sensitive and robust LC-MS platform for host cell protein characterization in biotherapeutics.

Host cell proteins (HCPs) are a major class of process-related impurities that need to be closely monitored during the production of biotherapeutics. Mass spectrometry (MS) has emerged as a promising tool for HCP analysis due to its specificity for individual HCP's identification and quantitation. However, utilization of MS as a routine characterization tool is still limited due to the time-consuming procedures, non-standardized instrumentation and methodologies, and the limited sensitivity compared to the enzyme-linked immunosorbent assays (ELISA). In this study, we introduced a sensitive (limit of detection (LOD) at 1-2 ppm) and robust HCP profiling platform method with suitable precision and accuracy that can be readily adopted to antibodies and other biotherapeutic modalities without the need for HCP enrichment. The NIST mAb and multiple in-house antibodies were analyzed, and results were benchmarked with other reported studies. In addition, a targeted analysis method with optimized sample preparation for absolute quantitation of lipases was developed and qualified with an LOD of 0.6 ppm and precision of <15%, which can be further improved to an LOD of 5 ppb by using the nano-flow LC.