Search Results (81)

Background: Recombinant monoclonal antibodies represent a vital category of biologics, constituting the largest class of molecules used to treat autoimmune disorders, cancers, rheumatoid arthritis, and other chronic conditions. The IgG1 subclass is the most potent among all the immunoglobulin gamma (IgG) antibodies, inducing Fc-related effector functions. N-linked glycan distribution of therapeutic IgG1s affects Fc-related effector functions such as CDC (complement-dependent cytotoxicity) and ADCC (antibody dependent cell-mediated cytotoxicity) biological activities and efficacy in vivo. Hence, as a critical quality attribute (CQA), the glycosylation profile of therapeutic IgG1s must be consistently preserved, which is primarily influenced by manufacturing process factors. In the era of biosimilars, it is challenging for biopharmaceutical manufacturers to not only obtain the desired glycan distribution consistently but also to meet the innovator molecule specifications as per the regulatory agencies. Methods: This study investigates the CHO fed-batch process parameters that affect the titer and terminal galactosylation of the TNF-α blocker-IgG1. It was hypothesized that galactose supplementation would enhance the galactosylation of TNF-α blocker-IgG1. Results: It was observed that such in-cultivation process shift does not affect cell culture parameters yet significantly enhances the galactosylation of TNF-α blocker-IgG1. Interestingly, the results indicate that supplementing D-galactose from the exponential phase of the CHO fed-batch process had the greatest effect on Fc galactosylation, increasing the amount of total galactosylated TNF-α blocker-IgG1 from 7.7% to 15.8%. Conclusions: Our results demonstrate a relatively easy and viable technique for cell culture engineering that is more appropriate for industrial production than costly in vitro glycoengineering. Full article

Public health crises triggered by viral infections pose severe threats to individual health and disrupt global socioeconomic systems. Against the backdrop of global pandemics caused by highly infectious diseases such as COVID-19 and Ebola virus disease (EVD), the development of innovative prevention and treatment strategies has become a strategic priority in the field of biomedicine. Neutralizing antibodies, as biological agents, are increasingly recognized for their potential in infectious disease control. Among these, nanobodies (Nbs) derived from camelid heavy-chain antibodies exhibit remarkable technical advantages due to their unique structural features. Compared to traditional neutralizing antibodies, nanobodies offer significant cost-effectiveness in production and enable versatile administration routes (e.g., subcutaneous injection, oral delivery, or aerosol inhalation), making them particularly suitable for respiratory infection control and resource-limited settings. Furthermore, engineered modification strategies—including multivalent constructs, multi-epitope recognition designs, and fragment crystallizable (Fc) domain fusion—effectively enhance their neutralizing activity and suppress viral immune escape mechanisms. Breakthroughs have been achieved in combating pathogens such as the Ebola virus and SARS-CoV-2, with mechanisms involving the blockade of virus–host interactions, induction of viral particle disintegration, and enhancement of immune responses. This review comprehensively discusses the structural characteristics, high-throughput screening technologies, and engineering strategies of nanobodies, providing theoretical foundations for the development of novel antiviral therapeutics. These advances hold strategic significance for addressing emerging and re-emerging infectious diseases. Full article

Antibody-based agents have become a preferred treatment for various diseases, including cancer, due to significant advances in antibody engineering. The use of single-chain Fv-Fcs (scFv-Fcs) has been a promising engineering approach for therapeutic design. The concept is that the Fc provides increased stability and target binding and ultimately improves performance. However, the structural and dynamic relationship between the variable and Fc domains, which are fused in close proximity, and the impact on stability and target binding are not well understood. This study evaluated trastuzumab-derived scFv-Fc antibodies, focusing on the impact of their design on important biopharmaceutical parameters. Computational modelling and molecular dynamics, alongside experimental studies, were used to ascertain their dynamics, expression and purification, stabilities, and binding potencies. The results showed that the scFv subunits exhibited stochastic interplays that lead to diverse shapes and were associated with functional performance. This new understanding of scFv-Fc antibodies and their structural and functional nuances provides important details to further guide the design of more effective and less toxic therapeutics. Full article

Allostery is a fundamental biological phenomenon that occurs when a molecule binds to a protein’s allosteric site, triggering conformational changes that regulate the protein’s activity. However, allostery in antibodies remains largely unexplored, and only a few reports have focused on allostery from antigen-binding fragments (Fab) to crystallizable fragments (Fc). But this study, using anti-phenobarbital antibodies—which are widely applied for detecting the potential health food adulterant phenobarbital—as a model and employing multiple computational methods, is the first to identify a cyclopeptide (cyclo[Link-M-WFRHY-K]) that induces allostery from Fc to Fab in antibody and elucidates the underlying antibody allostery mechanism. The combination of molecular docking and multiple allosteric site prediction algorithms in these methods identified that the cyclopeptide binds to the interface of heavy chain region-1 (CH₁) in antibody Fab and heavy chain region-2 (CH₂) in antibody Fc. Meanwhile, molecular dynamics simulations combined with other analytical methods demonstrated that cyclopeptide induces global conformational shifts in the antibody, which ultimately alter the Fab domain and enhance its antigen-binding activity from Fc to Fab. This result will enable cyclopeptides as a potential Fab-targeted allosteric modulator to provide a new strategy for the regulation of antigen-binding activity and contribute to the construction of novel immunoassays for food safety and other applications using allosteric antibodies as the core technology. Furthermore, graph theory analysis further revealed a common allosteric signaling pathway within the antibody, involving residues Q123, S207, S326, C455, A558, Q778, D838, R975, R1102, P1146, V1200, and K1286, which will be very important for the engineering design of the anti-phenobarbital antibodies and other highly homologous antibodies. Finally, the non-covalent interaction analysis showed that allostery from Fc to Fab primarily involves residue signal transduction driven by hydrogen bonds and hydrophobic interactions. Full article

Receptors for the immunoglobulin G constant fraction (FcγRs) are widely expressed in cells of the immune system. Complement-independent phagocytosis prompted FcγR research to show that the engagement of IgG immune complexes with FcγRs triggers a variety of cell host immune responses, such as phagocytosis, antibody-dependent cell cytotoxicity, and NETosis, among others. However, variants of these receptors have been implicated in the development of and susceptibility to autoimmune diseases such as systemic lupus erythematosus. Currently, the knowledge of FcγR variants is a required field of antibody therapeutics, which includes the engineering of recombinant soluble human Fc gamma receptors, enhancing the inhibitory and blocking the activating FcγRs function, vaccines, and organ transplantation. Importantly, recent interest in FcγRs is the antibody-dependent enhancement (ADE), a mechanism by which the pathogenesis of certain viral infections is enhanced. ADEs may be responsible for the severity of the SARS-CoV-2 infection. Therefore, FcγRs have become a current research topic. Therefore, this review briefly describes some of the historical knowledge about the FcγR type I family in humans, including the structure, affinity, and mechanism of ligand binding, FcγRs in diseases such as systemic lupus erythematosus (SLE), and the potential therapeutic approaches related to these receptors in SLE. Full article

Glioblastomas (GBMs) are lethal brain tumors in which EGFR gene amplification or mutation is frequently detected and is associated with poor prognosis. The standard of care is maximal resection followed by chemotherapy and radiation. Over the last twenty years, marginal improvements in patient survival have been achieved mainly through surgical techniques and the more accurate use of radiation. In this study, umbilical cord blood-derived and expanded human allogeneic natural killer (eNK) cells were pre-complexed to an Fc-engineered anti-EGFR monoclonal antibody (Pin-EGFR) to create Pin-EGFR-armed eNK cells. Pin-EGFR-armed eNK cells showed in vitro persistence of mAb anchoring. This arming process mediated specific, rapid and potent NK cell-redirected cytotoxicity against GBM cell lines and patient-derived cells in models consistent with the pathophysiological conditions of GBM. These results demonstrate the potential of Pin-EGFR-armed eNK cells to be an effective therapy against GBM cell lines in vitro. This product represents a promising strategy to directly target residual tumor tissue remaining at and beyond the resection margins immediately following GBM surgery to improve patient care. Full article

Background: Bispecific antibodies represent a promising class of biologics for cancer treatment. However, their dual specificity and complex structure pose challenges in the engineering process, often resulting in molecules with good functional but poor physicochemical properties. Method: To overcome limitations in the properties of an anti-5T4 x anti-CD3 (α5T4 x αCD3) DART molecule, a phage-display method was developed, which succeeded in simultaneously engineering cross-reactivity to the cynomolgus 5T4 ortholog, improving thermostability and the elevating expression level. Results: This approach generated multiple DART molecules that exhibited significant improvements in all three properties. The lead DART molecule demonstrated potent in vitro and in vivo anti-tumor activity. Although its clearance in human FcRn-transgenic mice was comparable to that of the parental molecule, faster clearance was observed in cynomolgus monkeys. The lead α5T4 x αCD3 DART molecule displayed no evidence of off-target binding or polyspecificity, suggesting that the increased affinity for the target may account for its accelerated clearance in cynomolgus monkeys. Conclusions: This may reflect target-mediated drug disposition (TMDD), a potential limitation of targeting 5T4, despite its limited expression in healthy tissues. Full article

This review briefly traces the historical development of antibody research and related technologies. The path from early perceptions of immunity to the emergence of modern immunotherapy has been marked by pivotal discoveries and technological advances. Early insights into immunity led to the development of vaccination and serotherapy. The elucidation of antibody structure and function paved the way for monoclonal antibody technology and its application in diagnosis and therapy. Breakthroughs in genetic engineering have enabled the production of humanized antibodies and the advances in Fc engineering, thereby increasing therapeutic efficacy. The discovery of immune checkpoints and cytokines revolutionized the treatment of cancer and autoimmune diseases. The field continues to evolve rapidly with the advent of antibody–drug conjugates, bispecific antibodies, and CAR T-cell therapies. As we face global health challenges, antibody research remains at the forefront of medical innovation and offers promising solutions for the future. Full article

The advent of recycling antibodies, leveraging pH-dependent antigen binding and optimized FcRn interaction, has advanced the field of antibody therapies, enabling extended durability and reduced dosages. Eculizumab (Soliris^®) demonstrated the efficacy of C5 inhibitors for paroxysmal nocturnal hemoglobinuria (PNH), while its derivative, ravulizumab (Ultomiris^®), recognized as a recycling antibody, extended the dosing intervals. However, limitations including intravenous administration and inefficacy in patients with the R885H single-nucleotide polymorphism (SNP) in C5 could necessitate alternative solutions. Crovalimab (PiaSky^®), a next-generation recycling antibody, overcomes these challenges with innovative charge engineering, achieving the enhanced cellular uptake of C5–crovalimab complexes and targeting a unique C5 epitope, allowing for efficacy regardless of the R885H SNP. This study highlights crovalimab’s distinctive molecular features, showing its eliminated binding to Fcγ receptors and C1q, alongside its optimized antigen binding characteristics. The impact of charge engineering was reconfirmed in mice, demonstrating faster C5 clearance than recycling antibodies. Notably, in the maintenance dosing regimen, crovalimab neutralizes approximately seven C5 molecules per antibody on average. Furthermore, its design also reduces the viscosity to facilitate high-concentration formulations suitable for subcutaneous delivery. Consequently, crovalimab offers a four-weekly subcutaneous injection regimen for PNH, marking a substantial improvement in treatment convenience and potentially transforming patients’ quality of life. Full article

Bispecific antibodies (BsAbs) are artificially engineered antibodies that can bind simultaneously to the CD3 subunit within the T-cell receptor complex and an antigen on tumor cells, leading to T-cell activation and tumor cell killing. BsAbs against BCMA or GPRC5D have shown impressive clinical activity in heavily pretreated patients with relapsed/refractory multiple myeloma (RRMM), with some agents having already received regulatory approval after the third (by the European Medicines Agency, EMA) or fourth (by the Food and Drug Administration, FDA) line of therapy; the results of early-phase clinical trials targeting FcRH5 are also promising. Overall, BsAbs as monotherapy correlated with an ORR that exceeded 60%, with a high CR rate ranging between 25% and 50% and a median PFS of around 1 year among patients with a median of 4–6 prior lines of therapy. The main toxicities include cytokine release syndrome, cytopenias, hypogammaglobulinemia, and infections; on-target off-tumor adverse events involving the skin, mucosa, hair, and nails may also occur with anti-GPRC5D BsAbs. Active research to increase their efficacy and improve their tolerance is still in progress, including combination therapies and application in earlier treatment lines and the development of novel agents. A better understanding of the mechanisms of resistance is a challenge and could lead to more personalized approaches. Full article

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further clinical development of these antibodies has been hampered by significant off-tumor toxicities. Here, we generated an anti-4-1BB/EGFR/PD-L1 trispecific antibody consisting of a triple-targeting tandem trimerbody (TT) fused to an engineered silent Fc region. This antibody (IgTT-4E1-S) was designed to combine the blockade of the PD-L1/PD-1 axis with conditional 4-1BB costimulation specifically confined to the tumor microenvironment (TME). The antibody demonstrated simultaneous binding to purified EGFR, PD-L1, and 4-1BB in solution, effective blockade of the PD-L1/PD1 interaction, and potent 4-1BB-mediated costimulation, but only in the presence of EGFR-expressing cells. These results demonstrate the feasibility of IgTT-4E1-S specifically blocking the PD-L1/PD-1 axis and inducing EGFR-conditional 4-1BB agonist activity. Full article

The vast majority of antibodies generated against a virus will be non-neutralising. However, this does not denote an absence of protective capacity. Yet, within the field, there is typically a large focus on antibodies capable of directly blocking infection (neutralising antibodies, NAbs) of either specific viral strains or multiple viral strains (broadly-neutralising antibodies, bNAbs). More recently, a focus on non-neutralising antibodies (nNAbs), or neutralisation-independent effects of NAbs, has emerged. These can have additive effects on protection or, in some cases, be a major correlate of protection. As their name suggests, nNAbs do not directly neutralise infection but instead, through their Fc domains, may mediate interaction with other immune effectors to induce clearance of viral particles or virally infected cells. nNAbs may also interrupt viral replication within infected cells. Developing technologies of antibody modification and functionalisation may lead to innovative biologics that harness the activities of nNAbs for antiviral prophylaxis and therapeutics. In this review, we discuss specific examples of nNAb actions in viral infections where they have known importance. We also discuss the potential detrimental effects of such responses. Finally, we explore new technologies for nNAb functionalisation to increase efficacy or introduce favourable characteristics for their therapeutic applications. Full article

Efforts to develop vaccine and immunotherapeutic countermeasures against the COVID-19 pandemic focus on targeting the trimeric spike (S) proteins of SARS-CoV-2. Vaccines and therapeutic design strategies must impart the characteristics of virion S from historical and emerging variants onto practical constructs such as soluble, stabilized trimers. The virus spike is a heterotrimer of two subunits: S1, which includes the receptor binding domain (RBD) that binds the cell surface receptor ACE2, and S2, which mediates membrane fusion. Previous studies suggest that the antigenic, structural, and functional characteristics of virion S may differ from current soluble surrogates. For example, it was reported that certain anti-glycan, HIV-1 neutralizing monoclonal antibodies bind soluble SARS-CoV-2 S but do not neutralize SARS-CoV-2 virions. In this study, we used single-molecule fluorescence correlation spectroscopy (FCS) under physiologically relevant conditions to examine the reactivity of broadly neutralizing and non-neutralizing anti-S human monoclonal antibodies (mAbs) isolated in 2020. Binding efficiency was assessed by FCS with soluble S trimers, pseudoviruses and inactivated wild-type virions representing variants emerging from 2020 to date. Anti-glycan mAbs were tested and compared. We find that both anti-S specific and anti-glycan mAbs exhibit variable but efficient binding to a range of stabilized, soluble trimers. Across mAbs, the efficiencies of soluble S binding were positively correlated with reactivity against inactivated virions but not pseudoviruses. Binding efficiencies with pseudoviruses were generally lower than with soluble S or inactivated virions. Among neutralizing mAbs, potency did not correlate with binding efficiencies on any target. No neutralizing activity was detected with anti-glycan antibodies. Notably, the virion S released from membranes by detergent treatment gained more efficient reactivity with anti-glycan, HIV-neutralizing antibodies but lost reactivity with all anti-S mAbs. Collectively, the FCS binding data suggest that virion surfaces present appreciable amounts of both functional and nonfunctional trimers, with neutralizing anti-S favoring the former structures and non-neutralizing anti-glycan mAbs binding the latter. S released from solubilized virions represents a nonfunctional structure bound by anti-glycan mAbs, while engineered soluble trimers present a composite structure that is broadly reactive with both mAb types. The detection of disparate antigenicity and immunoreactivity profiles in engineered and virion-associated S highlight the value of single-virus analyses in designing future antiviral strategies against SARS-CoV-2. Full article

The toolbox of modern antibody engineering allows the design of versatile novel functionalities exceeding nature’s repertoire. Many bispecific antibodies comprise heterodimeric Fc portions recently validated through the approval of several bispecific biotherapeutics. While heterodimerization methodologies have been established for low-throughput large-scale production, few approaches exist to overcome the bottleneck of large combinatorial screening efforts that are essential for the identification of the best possible bispecific antibody. This report presents a novel, robust and miniaturized heterodimerization process based on controlled Fab-arm exchange (cFAE), which is applicable to a variety of heterodimeric formats and compatible with automated high-throughput screens. Proof of applicability was shown for two therapeutic molecule classes and two relevant functional screening read-outs. First, the miniaturized production of biparatopic anti-c-MET antibody–drug conjugates served as a proof of concept for their applicability in cytotoxic screenings on tumor cells with different target expression levels. Second, the automated workflow enabled a large unbiased combinatorial screening of biparatopic antibodies and the identification of hits mediating potent c-MET degradation. The presented workflow utilizes standard equipment and may serve as a facile, efficient and robust method for the discovery of innovative therapeutic agents in many laboratories worldwide. Full article

The engineering of bispecific antibodies that exhibit optimal affinity and functional activity presents a significant scientific challenge. To tackle this, investigators employ an assortment of protein assay techniques, such as label-free interaction methodologies, which offer rapidity and convenience for the evaluation of extensive sample sets. These assays yield intricate data pertaining to the affinity towards target antigens and Fc-receptors, instrumental in predicting cellular test outcomes. Nevertheless, the fine-tuning of affinity is of paramount importance to mitigate potential adverse effects while maintaining efficient obstruction of ligand–receptor interactions. In this research, biolayer interferometry (BLI) was utilized to probe the functional characteristics of bispecific antibodies targeting cluster of differentiation 47 (CD47) and programmed death-ligand 1 (PD-L1) antigens, encompassing affinity, concurrent binding to two disparate antigens, and the inhibition of ligand–receptor interactions. The findings derived from BLI were juxtaposed with data from in vitro signal regulatory protein-α (SIRP-α)/CD47 blockade reporter bioassays for two leading bispecific antibody candidates, each demonstrating distinct affinity to CD47. Full article