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A basic FcRn-regulated clearance mechanism is investigated using the method of matched asymptotic expansions. The broader aim of the work is to obtain further insight on the mechanism, thereby providing theoretical support for future pharmacologically-based pharmacokinetic modelling efforts. The corresponding governing equations are first non-dimensionalised and the order of magnitudes of the model parameters are assessed based on their values reported in the literature. Under the assumption of high FcRn-binding affinity, analytical approximations are derived that are valid over the characteristic phases of the problem. Additionally, relatively simple equations relating clearance and AUC to physiological model parameters are derived, which are valid over the longest characteristic time scale of the problem. For lower to moderate doses clearance is effectively linear, whereas for higher doses it is nonlinear. It is shown that for all doses sufficiently high the leading-order approximation for the IgG concentration in plasma, over the longest characteristic time scale, is independent of the initial dose. This is because IgG that is in 'excess' of FcRn is eliminated over a time scale much shorter than that of the terminal phase. In conclusion, analytical approximations of the basic FcRn mechanism have been derived using matched asymptotic expansions, leading to a simple equation relating clearance to FcRn binding affinity, the ratio of degradation and FcRn concentration, and the volumes of the system. [ABSTRACT FROM AUTHOR]

Abstract Background Recently, several novel RSV immunisation products that protect infants and older adults against RSV disease have been licensed in Europe. We estimated the effectiveness and efficiency of introducing these RSV immunisation strategies in Germany. Methods We used a Bayesian framework to fit a deterministic age-structured dynamic transmission model of RSV to sentinel surveillance and RSV-specific hospitalisation data in Germany from 2015 to 2019. The calibrated model was used to evaluate different RSV intervention strategies over 5 years: long-acting, single-dose monoclonal antibodies (mAbs) in high-risk infants aged 1–5 months; long-acting mAbs in all infants aged 1–5 months; seasonal vaccination of pregnant women and one-time seasonal vaccination of older adults (75 + /65 + /55 + years). We performed sensitivity analysis on vaccine uptake, seasonal vs. year-round maternal vaccination, and the effect of under-ascertainment for older adults. Results The model was able to match the various RSV datasets. Replacing the current short-acting mAB for high-risk infants with long-acting mAbs prevented 1.1% of RSV-specific hospitalisations in infants per year at the same uptake. Expanding the long-acting mAB programme to all infants prevented 39.3% of infant hospitalisations per year. Maternal vaccination required a larger number to be immunised to prevent one additional hospitalisation than a long-acting mAB for the same uptake. Vaccination of adults older than 75 years at an uptake of 40% in addition to Nirsevimab in all infants prevented an additional 4.5% of all RSV hospitalisations over 5 years, with substantial uncertainty in the correction for under-ascertainment of the RSV burden. Conclusions Immunisation has the potential to reduce the RSV disease burden in Germany.

The COVID‐19 pandemic, driven by the SARS‐CoV‐2 virus, has posed a severe threat to global public health. Rapid, reliable, and easy‐to‐use detection methods for SARS‐CoV‐2 variants are critical for effective epidemic prevention and control. The N protein of SARS‐CoV‐2 serves as an ideal target for antigen detection. In this study, we achieved soluble expression of the recombinant SARS‐CoV‐2 N protein using an Escherichia coli expression system and generated specific monoclonal antibodies by immunizing BALB/c mice. We successfully developed 10 monoclonal antibodies against the N protein, designated 5B7, 5F2‐C11, 5E2‐E8, 6C3‐D8, 7C8, 9F2‐E9, 12H5‐D11, 13G2‐C10, 14E9‐F6, and 15H3‐E10. Using these antibodies, we established a sandwich ELISA with 6C3‐D8 as the capture antibody and 5F2‐C11 as the detection antibody. The assay demonstrated a sensitivity of 0.78 ng/mL and showed no cross‐reactivity with MERS‐CoV, HCoV‐OC43, HCoV‐NL63, and HCoV‐229E. Furthermore, this method successfully detected both wild‐type SARS‐CoV‐2 and its variants, including Alpha, Beta, Delta, and Omicron. These findings indicate that our sandwich ELISA exhibits excellent sensitivity, specificity, and broad‐spectrum applicability, providing a robust tool for detecting SARS‐CoV‐2 variants. [ABSTRACT FROM AUTHOR]

WuXiUPTM, WuXi Biologics' Ultra‐high Productivity platform, is an intensified and integrated continuous bioprocess platform developed for production of various biologics including monoclonal antibodies, fusion proteins, and bispecific antibodies. This process technology platform has manifested its remarkable capability in boosting the volumetric productivity of various biologics and has been implemented for large‐scale clinical material productions. In this paper, case studies of the production of different pharmaceutical proteins using two high‐producing and intensified culture modes of WuXiUPTM and the concentrated fed‐batch (CFB), as well as the traditional fed‐batch (TFB) are discussed from the perspectives of cell growth, productivity, and protein quality. Both WuXiUPTM and CFB outperformed TFB regarding volumetric productivity. Additionally, distinctive advantages in product quality profiles in the WuXiUPTM process, such as reduced acidic charge variants and fragmentation, are revealed. Therefore, a simplified downstream purification process with only two chromatographic steps can be developed to deliver the target product at a satisfactory purity and an extremely‐high yield. [ABSTRACT FROM AUTHOR]

Background: Recently, several novel RSV immunisation products that protect infants and older adults against RSV disease have been licensed in Europe. We estimated the effectiveness and efficiency of introducing these RSV immunisation strategies in Germany. Methods: We used a Bayesian framework to fit a deterministic age-structured dynamic transmission model of RSV to sentinel surveillance and RSV-specific hospitalisation data in Germany from 2015 to 2019. The calibrated model was used to evaluate different RSV intervention strategies over 5 years: long-acting, single-dose monoclonal antibodies (mAbs) in high-risk infants aged 1–5 months; long-acting mAbs in all infants aged 1–5 months; seasonal vaccination of pregnant women and one-time seasonal vaccination of older adults (75 + /65 + /55 + years). We performed sensitivity analysis on vaccine uptake, seasonal vs. year-round maternal vaccination, and the effect of under-ascertainment for older adults. Results: The model was able to match the various RSV datasets. Replacing the current short-acting mAB for high-risk infants with long-acting mAbs prevented 1.1% of RSV-specific hospitalisations in infants per year at the same uptake. Expanding the long-acting mAB programme to all infants prevented 39.3% of infant hospitalisations per year. Maternal vaccination required a larger number to be immunised to prevent one additional hospitalisation than a long-acting mAB for the same uptake. Vaccination of adults older than 75 years at an uptake of 40% in addition to Nirsevimab in all infants prevented an additional 4.5% of all RSV hospitalisations over 5 years, with substantial uncertainty in the correction for under-ascertainment of the RSV burden. Conclusions: Immunisation has the potential to reduce the RSV disease burden in Germany. [ABSTRACT FROM AUTHOR]

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up (UHMU)-level ultra-high-performance liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis to improve N-glycan profiling of mAbs. Two specific enzymes, known as IgG degradation enzymes (IGDEs), were used to selectively cleave therapeutic mAbs above the hinge region to separate antibody subunits for further Fc glycan analysis by means of the UHMU/LC-HRMS workflow. The complexity of the mass spectra of IGDEs-digested mAbs was significantly reduced compared to the intact MS level, enabling reliable assignment and relative quantitation of paired Fc glycoforms. The results of the UHMU/LC-HRMS analysis of nine approved therapeutics highlight the significance of this approach for in-depth glycoform profiling. [ABSTRACT FROM AUTHOR]

Bispecific antibodies (bsAbs) are a class of antibodies that can mediate novel mechanisms of action compared to monospecific monoclonal antibodies (mAbs). Since the discovery of mAbs and their adoption as therapeutic agents in the 1980s and 1990s, the development of bsAbs has held substantial appeal. Nevertheless, only three bsAbs (catumaxomab, blinatumomab, emicizumab) were approved through the end of 2020. However, since then, 11 bsAbs received regulatory agency approvals, of which nine (amivantamab, tebentafusp, mosunetuzumab, cadonilimab, teclistamab, glofitamab, epcoritamab, talquetamab, elranatamab) were approved for the treatment of cancer and two (faricimab, ozoralizumab) in non-oncology indications. Notably, of the 13 currently approved bsAbs, two, emicizumab and faricimab, have achieved blockbuster status, showing the promise of this novel class of therapeutics. In the 2020s, the approval of additional bsAbs can be expected in hematological malignancies, solid tumors and non-oncology indications, establishing bsAbs as essential part of the therapeutic armamentarium.

The essential interaction between Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) and basigin makes RH5 a prime target for broadly neutralizing antibodies. A recent study by Barrett et al. mapped the RH5 antigenic landscape from RH5.1/AS01 B vaccinees and identified a potent public antibody clonotype, advancing malaria vaccine and prophylactic antibody development. [ABSTRACT FROM AUTHOR]

An important virulence factor in the pathogenesis of infections caused by Pseudomonas aeruginosa is flagellin: it serves as the main structural component of the bacterial flagellum and an acceptor for the TLR5 receptor of the innate immune system. Toll-like receptor 5 is able to bind bacterial flagellin and activate the anti-inflammatory transcription factor NF-kB through the adapter protein MyD88, which induces the production of anti-inflammatory cytokines. The inclusion of flagellin in recombinant proteins increased the ability to stimulate the production of anti-inflammatory cytokines and activate antigen-presenting cells. A number of experiments have shown that the use of flagellin as a molecular adjuvant in vaccines increases the expression of CD80, CD83, CD86 and MHC II molecules on the surface of dendritic cells, and also leads to an increase in the secretion of IFNγ and α-defensins by dendritic and NK cells; T cell proliferation and activation of antigen-specific cytotoxic T lymphocytes, as well as increased induction of antigen-specific IgG and IgA antibodies. Due to the natural and acquired resistance of P. aeruginosa to antibiotics, the available choice of antipseudomonas drugs is decreasing, and therefore the problem of developing effective therapeutic drugs to protect against this infection is of high medical and social importance. For this purpose, it seems promising to study the immunobiological properties of P. aeruginosa flagellin as a possible vaccine component. Based on this, in the Laboratory of Protective Antigens of the I. Mechnikov Research Institute of Vaccines and Sera, recombinant flagellin C (FliC) of P. aeruginosa was obtained, and its immunogenicity and protective properties were proven. However, the question of standardizing methods for screening and monitoring the resulting recombinant FliC protein remains open. To solve this issue, hybridomas producing monoclonal antibodies (mAb) of a given specificity were obtained, the basic immunochemical properties of mAbs were studied, and the possibility of using them as reagents in constructing a test system for identifying and standardizing the recombinant FliC protein upon its production was assessed. Purpose of the work: to obtain monoclonal antibodies to the recombinant flagellin C protein of P. aeruginosa; to study their basic immunochemical properties and to evaluate the possibility of using the recombinant FliC protein for screening and control.

Abstract The initial Phase-I single centre, single dose, randomized, double-blind, cross-over study was planned to assess the pharmacokinetic and pharmacodynamic bioequivalence of the trastuzumab biosimilar (MYL-1401O) compared to the reference Herceptin®. Their respective immunomodulation profile presented in this paper involved healthy males receiving a single infusion of both monoclonals, separated by a washout period. Sixty parameters were assessed in total, including serum cytokines, peripheral mononuclear cell (PBMC) subsets, cell activation and response to recall antigens and mitogen, pre- and post- infusion, as well as a cytokine release assay (CRA) at baseline. Trastuzumab infusion induced a transient and weak peak of serum IL-6 at 6 h, and a modulation of mononuclear cell subset profile and activation level, notably CD16 + cells. Except for CD8 + T cells, there were no significant differences between Herceptin® and MYL-1401O. In CRA, PBMC stimulated with MYL-1401O or Herceptin® similarly secreted IL-6, TNF-α, IL-1β, GM-CSF, IFN-γ, and IL-10, but no or low level of IL-2. Interestingly, some observed adverse events correlated with IL-2 and IFN-γ in CRA. MYL-1401O exhibited a very similar immunomodulation profile to Herceptin®, strongly supporting its bioequivalence. This approach may thus be included in a proof-of-concept study. CRA may be used as a predictive assay for the evaluation of clinical monoclonals.

The significant increase in product titers, coupled with the growing focus on continuous bioprocessing, has renewed interest in using precipitation as a low‐cost alternative to Protein A chromatography for the primary capture of monoclonal antibody (mAb) products. In this work, a commercially relevant mAb was purified from clarified cell culture fluid using a tubular flow precipitation reactor with dewatering and washing provided by tangential flow microfiltration. The particle morphology was evaluated using an inline high‐resolution optical probe, providing quantitative data on the particle size distribution throughout the precipitation process. Data were obtained in both a lab‐built 2‐stage countercurrent washing system and a commercial countercurrent contacting skid that provided 4 stages of continuous washing. The processes were operated continuously for 2 h with overall mAb yield of 92 ± 3% and DNA removal of nearly 3 logs in the 4‐stage system. The high DNA clearance was achieved by selective redissolution of the mAb using a low pH acetate buffer. Host cell protein clearance was 0.59 ± 0.08 logs, comparable to that based on model predictions. The process mass intensity was slightly better than typical Protein A processes and could be significantly improved by preconcentration of the antibody feed material. [ABSTRACT FROM AUTHOR]

In the scenario of an integrated space–air–ground emergency communication network, users encounter the challenge of rapidly identifying the optimal network node amidst the uncertainty and stochastic fluctuations of network states. This study introduces a Multi-Armed Bandit (MAB) model and proposes an optimization algorithm leveraging dynamic variance sampling (DVS). The algorithm posits that the prior distribution of each node's network state conforms to a normal distribution, and by constructing the distribution's expected value and variance, it maximizes the utilization of sample data, thereby maintaining an equilibrium between data exploitation and the exploration of the unknown. Theoretical substantiation is provided to illustrate that the Bayesian regret associated with the algorithm exhibits sublinear growth. Empirical simulations corroborate that the algorithm in question outperforms traditional ε-greedy, Upper Confidence Bound (UCB), and Thompson sampling algorithms in terms of higher cumulative rewards, diminished total regret, accelerated convergence rates, and enhanced system throughput. [ABSTRACT FROM AUTHOR]

Objective: Provide clinicians with current evidence for biologic therapy in children with chronic rhinosinusitis with nasal polyposis (CRSwNP). Data Sources: PubMed, MEDLINE, Cochrane, and clinical trial registries. Review Methods: Key search terms related to biologic therapy in pediatric CRSwNP were identified via a structured query of current medical literature and clinical trial databases. Conclusions: There is a dearth of active clinical trials and research studies for biologics targeting pediatric CRSwNP. There is an ongoing compassionate‐use clinical trial involving Dupilumab for children with nasal polyps as well as only 1 published work specifically focused on Dupilumab for pediatric CRSwNP in the setting of aspirin‐exacerbated respiratory disease. Implications for Practice: For children with atopic dermatitis, asthma, and chronic idiopathic urticaria, biologic therapies such as Omalizumab, Dupilumab, and Mepolizumab have gained Food and Drug Administration approval. The role of biologic therapy in pediatric CRSwNP demonstrates significant promise in the comprehensive management of the unified airway. Additional Phase III trials are necessary to broaden clinical indications for children with comorbid conditions and complex sinonasal disease. [ABSTRACT FROM AUTHOR]

Background: The introduction of biological drugs in the management of chronic rhinosinusitis with nasal polyps (CRSwNP) is allowing new and increasingly promising therapeutic options. This manuscript aims to provide a multicenter trial in a real-life setting on Mepolizumab treatment for severe uncontrolled CRSwNP with or without comorbid asthma. Methods: A retrospective data analysis was jointly conducted at the Otolaryngology–Head and Neck Surgery departments of La Sapienza University and San Camillo Forlanini Hospital in Rome. Both institutions participated by sharing clinical information on patients with CRSwNP treated with Mepolizumab. Patients were evaluated before starting Mepolizumab, at six months and at twelve months from the first drug administration. During follow–up visits, patients underwent endoscopic evaluation, quality of life assessment, nasal symptoms assessment, and blood tests to monitor mainly neutrophils, basophils, eosinophils, and IgG, IgA, and IgE assay. Results: Twenty patients affected by CRSwNP and treated with Mepolizumab were enrolled (12 females and 8 males with a mean age of 63.7 years). Sixteen patients (80%) had concomitant asthma. During follow-up, a gradual improvement in nasal polyp score, quality of life and nasal symptoms, assessed by SNOT-22 and VAS and loss of smell measured by olfactory VAS, was found. Regarding blood tests, eosinophils decreased gradually, while other blood parameters showed no statistically significant changes. Conclusions: Mepolizumab has been shown to be effective in the therapeutic management of patients with CRSwNP. Further studies are needed to support our findings and better understand the underlying immune pathways to predict patients' response to biological treatment in CRSwNP. [ABSTRACT FROM AUTHOR]

Through machine learning, identifying correlations between amino acid sequences of antibodies and their observed characteristics, we developed an internal viscosity prediction model to empower the rapid engineering of therapeutic antibody candidates. For a highly viscous anti-IL-13 monoclonal antibody, we used a structure-based rational design strategy to generate a list of variants that were hypothesized to mitigate viscosity. Our viscosity prediction tool was then used as a screen to cull virtually engineered variants with a probability of high viscosity while advancing those with a probability of low viscosity to production and testing. By combining the rational design engineering strategy with the in silico viscosity prediction screening step, we were able to efficiently improve the highly viscous anti-IL-13 candidate, successfully decreasing the viscosity at 150 mg/mL from 34 cP to 13 cP in a panel of 16 variants. [ABSTRACT FROM AUTHOR]

Host cell protein (HCP) characterization is a crucial quality parameter for biotherapeutic drug safety and stability. With a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach, we identified ubiquitin in ultrafiltration/diafiltration (UF/DF) pools of one of our monoclonal antibody (mAb) products. Since ubiquitin occurs physiologically as a post-translational modification (PTM) involved in many cellular functions, we suspected the possibility that if identified as an HCP, it may occur as a covalent modification on the mAb. In fact, in this study we characterized and quantified the ubiquitin modification on the Fc domain of mAbX by data dependent acquisition (DDA) and data independent acquisition (DIA) – MS workflows. Covalent binding and site localization were confirmed by identifying a characteristic diglycine motif on the modified peptide. Initially observed reduced detectability of ubiquitin in samples prepared with native digestion was attributed to impaired digestion and subsequent removal along with the mAb in the precipitation step. Our work has contributed to a better understanding of ubiquitin as an HCP considering its specific features such as occurrence in different topologies and provided insight into how covalent binding to a drug product can affect its identification by MS when native digestion conditions are used. [ABSTRACT FROM AUTHOR]

Monitoring the genetic variability of human respiratory syncytial virus (hRSV) is of paramount importance, especially for the potential implication of key antigenic mutations on the emergence of immune escape variants. Thus, to describe the genetic diversity and evolutionary dynamics of hRSV circulating in Sicily (Italy), a total of 153 hRSV whole-genome sequences collected from 770 hRSV-positive subjects between 2017 and 2023, before the introduction of expanded immunization programs into the population, were investigated. The phylogenetic analyses indicated that the genotypes GA.2.3.5 (ON1) for hRSV-A and GB.5.0.5a (BA9) for hRSV-B co-circulated in our region. Amino acid (AA) substitutions in the surface and internal proteins were evaluated, including the F protein antigenic sites, as the major targets of immunoprophylactic monoclonal antibodies and vaccines. Overall, the proportion of AA changes ranged between 1.5% and 22.6% among hRSV-A, whereas hRSV-B varied in the range 0.8–16.9%; the latter was more polymorphic than hRSV-A within the key antigenic sites. No AA substitutions were found at site III of both subgroups. Although several non-synonymous mutations were found, none of the polymorphisms known to potentially affect the efficacy of current preventive measures were documented. These findings provide new insights into the global hRSV molecular epidemiology and highlight the importance of defining a baseline genomic picture to monitor for future changes that might be induced by the selective pressures of immunological preventive measures, which will soon become widely available. [ABSTRACT FROM AUTHOR]

The initial Phase-I single centre, single dose, randomized, double-blind, cross-over study was planned to assess the pharmacokinetic and pharmacodynamic bioequivalence of the trastuzumab biosimilar (MYL-1401O) compared to the reference Herceptin®. Their respective immunomodulation profile presented in this paper involved healthy males receiving a single infusion of both monoclonals, separated by a washout period. Sixty parameters were assessed in total, including serum cytokines, peripheral mononuclear cell (PBMC) subsets, cell activation and response to recall antigens and mitogen, pre- and post- infusion, as well as a cytokine release assay (CRA) at baseline. Trastuzumab infusion induced a transient and weak peak of serum IL-6 at 6 h, and a modulation of mononuclear cell subset profile and activation level, notably CD16 + cells. Except for CD8 + T cells, there were no significant differences between Herceptin® and MYL-1401O. In CRA, PBMC stimulated with MYL-1401O or Herceptin® similarly secreted IL-6, TNF-α, IL-1β, GM-CSF, IFN-γ, and IL-10, but no or low level of IL-2. Interestingly, some observed adverse events correlated with IL-2 and IFN-γ in CRA. MYL-1401O exhibited a very similar immunomodulation profile to Herceptin®, strongly supporting its bioequivalence. This approach may thus be included in a proof-of-concept study. CRA may be used as a predictive assay for the evaluation of clinical monoclonals. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13C′, 13Cα, 13Cβ resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs' higher order structures, dynamics and interactions, using solution-state NMR. [ABSTRACT FROM AUTHOR]

The respiratory syncytial virus (RSV) causes significant respiratory disease in young infants and the elderly. Immune prophylaxis in infants is currently limited to palivizumab, an anti-RSV fusion (F) protein monoclonal antibody (mAb). While anti-F protein mAbs neutralize RSV, they are unable to prevent aberrant pathogenic responses provoked by the RSV attachment (G) protein. Recently, the co-crystal structures of two high-affinity anti-G protein mAbs that bind the central conserved domain (CCD) at distinct non-overlapping epitopes were solved. mAbs 3D3 and 2D10 are broadly neutralizing and block G protein CX3C-mediated chemotaxis by binding antigenic sites γ1 and γ2, respectively, which is known to reduce RSV disease. Previous studies have established 3D3 as a potential immunoprophylactic and therapeutic; however, there has been no similar evaluation of 2D10 available. Here, we sought to determine the differences in neutralization and immunity to RSV Line19F infection which recapitulates human RSV infection in mouse models making it useful for therapeutic antibody studies. Prophylactic (24 h prior to infection) or therapeutic (72 h post-infection) treatment of mice with 3D3, 2D10, or palivizumab were compared to isotype control antibody treatment. The results show that 2D10 can neutralize RSV Line19F both prophylactically and therapeutically, and can reduce disease-causing immune responses in a prophylactic but not therapeutic context. In contrast, 3D3 was able to significantly (p < 0.05) reduce lung virus titers and IL-13 in a prophylactic and therapeutic regimen suggesting subtle but important differences in immune responses to RSV infection with mAbs that bind distinct epitopes.

SCIENTIFIC RELEVANCE. An important quality-control issue for therapeutic monoclonal antibodies (mAbs) is the determination of residual protein A leaching from the carrier during the purification of mAbs by affinity chromatography. However, unrelated compounds (immunoglobulins) present in the sample can complicate the immunoenzymatic detection of protein A (matrix effect), potentially leading to false-negative test results. To increase the efficiency of enzyme-linked immunosorbent assay (ELISA), it is necessary to develop a sample preparation step that can irreversibly break the bond in the protein A–mAb complex.AIM. This study aimed to develop and validate an analytical procedure for the determination of residual protein A in active substances of therapeutic mAbs by ELISA with a test kit comprising in-house reagents.MATERIALS AND METHODS. Recombinant protein A was used as an antigen. Polyclonal antibodies to protein A were produced by immunising chickens, selecting immunised eggs, and isolating antibodies from these eggs. Protein A-specific antibodies were purified by affinity chromatography. Residual protein A was determined using sandwich ELISA with preliminary sample preparation. The analytical procedure was validated in accordance with the requirements of the State Pharmacopoeia of the Russian Federation (Validation of Analytical Procedures, OFS.1.1.0012).RESULTS. The authors obtained, isolated, and purified chicken IgY antibodies to recombinant protein A. The authors selected sample preparation conditions for the determination of residual protein A by ELISA and optimum compositions of buffer solutions, including the composition of a denaturing buffer to disrupt the protein A–mAb complex. The developed analytical procedure was validated. According to the measurements of protein A, the accuracy of the analytical procedure ranged within 83–108% of the nominal value, the interlaboratory precision ranged within 96–116%, and the repeatability was up to 13%. The lower limit of quantitation was 10 ng/mL with a minimum required dilution of 1:10. The analytical range extended from 10 to 40 ng/mL. The analytical procedure showed results comparable to those obtained with a similar test kit from an international manufacturer.CONCLUSIONS. The developed analytical procedure for the determination of residual protein A by ELISA with a test kit comprising in-house reagents can minimise the matrix effect in therapeutic mAbs. This analytical procedure will alleviate import substitution and reduce quality control costs for Russian immunobiologicals.

Purpose: Silicone oil droplets in biopharmaceutical products can originate from sources such as siliconized surfaces of primary packaging materials, potentially triggering the formation of protein–silicone oil particles. To better understand this phenomenon, there is a need for particle detection devices that cannot only distinguish between protein particles and silicone oil droplets but also determine particle sizes ranging from nanometers to micrometers. Method: In this study, we conducted a systematic assessment of imaging flow cytometry (IFC) using the FlowSight® instrument. Our first step was to investigate specific instrument settings using protein particle samples spiked with silicone oil for particle classification. Based on these findings, we established suitable, harmonized working templates. Next, we evaluated the instrument’s accuracy and precision for particle sizes within the range of 0.5 to 100 µm and their respective concentrations. Finally, we investigated any constraints in particle concentration within this size range. Results: This study demonstrates that IFC can effectively distinguish protein particles from silicone oil droplets when the latter is labeled with a specific fluorescent dye. Our findings suggest that fluorescently labeled particles ≥ 0.5 µm can be reliably detected. Through our research, we determined the particle concentration limits for each particle size in the range of 0.5 to 10 µm, with a precision deviation of less than 15%. However, our study also revealed that IFC exhibited insufficient accuracy for the tested particle concentrations within this size range. Additionally, we showed that the measurements were significantly influenced by the instrument settings. Conclusion: Although we addressed numerous new aspects to enhance the experimental procedure of IFC measurements, we conclude that IFC is not an ideal technique for quantifying sub-visible particles. Instead, it should be employed to provide supportive characterization data in conjunction with commonly used sub-visible particle detection methods. If distinguishing between protein particles and silicone oil droplets is essential, IFC is an option, as long as the fluorescent dye is carefully selected. [ABSTRACT FROM AUTHOR]

• Thermal stability of polysorbate-degradative enzymes was assessed with chemical proteomics and biophysical studies. • Brief heat treatment inactivated polysorbate-degradative enzymes and had minimal impact on the therapeutic antibody. • Heat inactivation of protein-A pool decelerated polysorbate degradation. Polysorbate degradation in biotherapeutics formulations is an industry-wide problem and mainly caused by residual host cell-derived enzymes. We present a proof-of-concept study of a control strategy which takes advantage of lower thermal stability of such enzymes relative to therapeutic proteins. We profiled heat sensitivity of host cell–derived enzyme activity with chemical proteomics and observed that PLA2G7 became inactive after brief heating. Further biophysical studies indicated that these enzymes were less thermally stable than a monoclonal antibody. Importantly, brief heat treatment had minimal impact on the stability of the antibody. Consequently, heat inactivation of polysorbate-spiked protein-A pool decelerated polysorbate degradation. This study suggests that heat inactivation of host cell–derived enzymes could be a control stragy for polysorbate degradation. [ABSTRACT FROM AUTHOR]

The use of human antibodies as biologic therapeutics has revolutionized patient care throughout fields of medicine. As our understanding of the many roles antibodies play within our natural immune responses continues to advance, so will the number of therapeutic indications for which an mAb will be developed. The great breadth of function, long half-life, and modular structure allow for nearly limitless therapeutic possibilities. Human antibodies can be rationally engineered to enhance their desired immune functions and eliminate those that may result in unwanted effects. Antibody therapeutics now often start with fully human variable regions, either acquired from genetically engineered humanized mice or from the actual human B cells. These variable genes can be further engineered by widely used methods for optimization of their specificity through affinity maturation, random mutagenesis, targeted mutagenesis, and use of in silico approaches. Antibody isotype selection and deliberate mutations are also used to improve efficacy and tolerability by purposeful fine-tuning of their immune effector functions. Finally, improvements directed at binding to the neonatal Fc receptor can endow therapeutic antibodies with unbelievable extensions in their circulating half-life. The future of engineered antibody therapeutics is bright, with the global mAb market projected to exhibit compound annual growth, forecasted to reach a revenue of nearly half a trillion dollars in 2030. [ABSTRACT FROM AUTHOR]

Extracellular domain (ECD) antigens are crucial components for antibody discovery, in vitro assays, and epitope mapping during therapeutical antibody development. Oftentimes, those antigens are difficult to produce while retaining the biologic function/activity upon extracellular secretion in commonly used expression systems. We have developed an effective method to cope with the challenge of generating quality antigen ECDs. In this method, a monoclonal antibody (Mab) or antibody fragment antigen‐binding (Fab) region acts as a "chaperone" to stabilize the antigen ECD through forming an antibody:antigen complex. This methodology includes transient co‐expression of the complex in Chinese hamster ovary cells and then dissociation of the purified complex into individual components by low pH treatment in the presence of arginine. The antigen is then separated from the chaperone on a preparative size exclusion chromatography (pSEC) followed by an optional affinity chromatography process to remove residual Mab or Fab. We demonstrate this co‐expression/disassociation methodology on two difficult‐to‐express antigen ECDs from cluster‐of‐differentiation/cytokine family and were successful in producing stable, biologically active antigens when the common methods using Histidine‐tagged and/or Fc‐fused protein failed. This can be applied as a general approach for antigen production if a Mab or binding partner is available. [ABSTRACT FROM AUTHOR]

ABSTRACT With some exceptions, global policymakers have recommended against the use of existing monoclonal antibodies in COVID-19 due to loss of neutralization of newer variants. The purpose of this study was to investigate the impact of Ronapreve on compartmental viral replication using paradigms for susceptible and insusceptible variants. Virological efficacy and impact on pathogenicity was assessed in K18-hACE2 mice inoculated with either the Delta or BA.1 Omicron variants. Ronapreve reduced sub-genomic viral RNA levels in lung and nasal turbinate, 4 and 6 days post-infection, for the Delta variant but not the Omicron variant. It also blocked brain infection, which is seen with high frequency in K18-hACE2 mice after Delta variant infection. At day 6, the inflammatory response to lung infection with the Delta variant was altered to a multifocal granulomatous inflammation in which the virus appeared to be confined. The current study provides evidence of an altered tissue response to SARS-CoV-2 after treatment with a monoclonal antibody combination that retains neutralization activity. These data demonstrate that experimental designs that reflect treatment use cases are achievable in animal models for monoclonal antibodies. Extreme caution should be taken when interpreting prophylactic experimental designs that may not be representative of treatment.IMPORTANCEFollowing the emergence of the SARS-CoV-2 Omicron variant, the WHO recommended against the use of Ronapreve in its COVID-19 treatment guidelines due to a lack of efficacy based on current pharmacokinetic-pharmacodynamic understanding. However, the continued use of Ronapreve, specifically in vulnerable patients, was advocated by some based on in vitro neutralization data. Here, the virological efficacy of Ronapreve was demonstrated in both the lung and brain compartments using Delta as a paradigm for a susceptible variant. Conversely, a lack of virological efficacy was demonstrated for the Omicron variant. Comparable concentrations of both monoclonal antibodies were observed in the plasma of Delta- and Omicron-infected mice. This study made use of a reliable murine model for SARS-CoV-2 infection, an experimental design reflective of treatment, and demonstrated the utility of this approach when assessing the effectiveness of monoclonal antibodies.

Background: Tixagevimab-cilgavimab is a combination of 2 mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In December 2021, the Food and Drug Administration issued Emergency Use Authorization for intramuscular injection of tixagevimab-cilgavimab for prophylaxis against SARS-CoV-2 in immunocompromised patients. Shortly thereafter, our clinic distributed tixagevimab-cilgavimab to patients with common variable immunodeficiency. Objective: We sought to evaluate the effectiveness and tolerability of tixagevimab-cilgavimab in a common variable immunodeficiency clinic. Methods: A retrospective chart review from February 1, 2022, to August 1, 2022, of 47 patients with common variable immunodeficiency who were offered tixagevimab-cilgavimab was carried out. Comparative outcomes of treatment and nontreatment groups examined the occurrence of SARS-CoV-2 infection, severity of SARS-CoV-2 infection, and other non–SARS-CoV-2 infections. Results: Seventy percent of the patients were female; mean age was 49 years. Twenty-three patients received tixagevimab-cilgavimab, and 24 did not receive prophylaxis. In the tixagevimab-cilgavimab group, all were vaccinated for SARS-CoV-2 and 22 were receiving immunoglobulin replacement. One patient was infected with SARS-CoV-2, no patients required emergency care, and 7 patients had non–SARS-CoV-2 infection. In the cohort that did not receive prophylaxis, 21 were vaccinated, and all received immunoglobulin replacement. Two patients tested positive for SARS-CoV-2, 1 patient required emergency care due to SARS-CoV-2 disease severity, and 4 patients had a non–SARS-CoV-2 infection. None of the results showed statistical significance. Conclusions: Although there is evidence that tixagevimab-cilgavimab can be protective against SARS-CoV-2 in immunocompromised individuals, our data suggest that this benefit may be blunted in patients with common variable immunodeficiency on immunoglobulin replacement. The additional benefit of tixagevimab-cilgavimab in immunocompromised patients already receiving replacement therapy requires further exploration.

ABSTRACT Influenza A virus (IAV) is well known for its pandemic potential. While current surveillance and vaccination strategies are highly effective, therapeutic approaches are often short-lived due to the high mutation rates of IAV. Recently, monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach, both against current strains and future IAV pandemics. In addition to mAbs, several antibody-like alternatives exist, which aim to improve upon mAbs. Among these, Affimers stand out for their short development time, high expression levels in Escherichia coli, and animal-free production. In this study, we utilized the Affimer platform to isolate and produce specific and potent inhibitors of IAV. Using a monomeric version of the IAV trimeric hemagglutinin (HA) fusion protein, we isolated 12 Affimers that inhibit IAV infection in vitro. Two of these Affimers were characterized in detail and exhibited nanomolar-binding affinities to the target H3 HA protein, specifically binding to the HA1 head domain. Cryo-electron microscopy (cryo-EM), employing a novel spray approach to prepare cryo-grids, allowed us to image HA-Affimer complexes. Combined with functional assays, we determined that these Affimers inhibit IAV by blocking the interaction of HA with the host-cell receptor, sialic acid. Furthermore, these Affimers inhibited IAV strains closely related to the one used for their isolation. Overall, our results support the use of Affimers as a viable alternative to existing targeted therapies for IAV and highlight their potential as diagnostic reagents.IMPORTANCEInfluenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro. We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo, administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

Abstract Introduction Treatment with monoclonal antibodies provides rapid, passive immunity and may stop COVID-19 disease progression. The study evaluated the effect of bamlanivimab (BAM) or BAM + etesevimab (ETE)/sotrovimab compared to placebo on SARS-CoV-2 viral load in patients with COVID-19. Methods The phase 2, randomized, single-dose study included patients aged between ≥ 18 and 5.27 on day 7 (persistently high viral load [PHVL]) who received BAM or BAM + (ETE or sotrovimab). Results A total of 725 patients, mean age 39.6 years (range 18–75 years), 50.2% male were randomized and infused with study drug in arms 1–6; and a total 202 patients, mean age 38 years (range 18–63 years), 53.5% female were randomized and infused with study drug in arms 7 and 8. A significantly lower proportion of patients in arms 2–6 and arm 7 experienced PHVL on day 7 compared to placebo. On day 7, patients in arms 2, 3, and 6 consistently experienced significantly greater reduction in viral load than placebo. Significant improvement was observed in time to viral load clearance and time to symptom improvement by day 29 in some arms compared to placebo. No new safety concerns were observed with drug combinations. Conclusion The study demonstrated that a significantly lower proportion of patients with mild-to-moderate COVID-19 treated with BAM or BAM + (ETE or sotrovimab) experienced a PHVL at day 7. Trial Registration ClinicalTrials.gov identifier, NCT04634409.

Abstract Background Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. Methods An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. Results A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M−1 and 2.08 × 107 M−1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. Conclusion The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics.

Abstract H9N2 avian influenza viruses (AIVs) are widely distributed, causing continuous outbreaks in poultry and sporadic infections in humans. Vaccination is the primary method used to prevent and control H9N2 AIV infection. However, the ongoing evolution and mutation of AIVs often result in limited protection effects from vaccines. Therapeutic monoclonal antibodies (mAbs) targeting influenza viruses offer a promising alternative. In this study, we immunized mice with inactivated H9N2-W1 virus, and we screened and acquired five mAbs, namely 4D12, F4, 5C8, 2G8 and A11. We showed that all five mAbs specifically targeted the HA protein of various H9N2 AIV strains. In vitro neutralization tests demonstrated that all five mAbs exhibited neutralization activity against H9N2 AIVs, with mAb F4 displaying the most potent neutralization effect. The F4 mAb exhibited dose-dependent preventive and therapeutic effects against lethal H9N2-115 infection, and the administration of F4 at a dose of 3 μg/g provided complete protection in vivo. Our study presents an alternative approach for preventing and controlling H9N2 AIV infection. Furthermore, the identified F4 mAb holds promise as a solution to potential pandemics in humans caused by H9N2 AIVs.

Investigating products under stress conditions provides valuable information for assessing stability, biosimilarity, and degradation behaviors during monoclonal antibody (mAb) development. Proper sample preparation is crucial for accurately evaluating the biosimilarity and effects of stress conditions in comparability assessment, where these studies guide biosimilar mAb development steps. Enzymatic and chemical treatments applied during sample preparation of mAbs generally require treatment of samples in temperatures higher than the storage temperatures of antibodies.In this study, samples of a TNF-α inhibitor IgG1 biosimilar (BIO) and its originator (OR) were treated for 7 days at commonly used temperatures during sample preparation. Alterations in the intact IgG, size variants, charge variants, binding kinetics, and post-translational modifications (PTMs) were investigated with CE-SDS, SE-UPLC, icIEF, SPR, and LC-MS/MS, respectively. Samples treated at 50 °C exhibited significant degradation, while minor differences were observed in samples treated at 37 °C. Monomer and intact IgG levels were decreased to levels below 97 % and 94 %, respectively, after 7 days of thermal treatment at 50 °C for both BIO and OR samples. Similar rates of degradation were observed between the treated biosimilar and originator samples. The percent monomer degradation rate between the biosimilar and the originators was similar at 50 °C (p = 0.32). Thermal treatment increased acidic variant levels in the products of the BIO (23.10 %) and OR (23.16 %). During post-translational modification monitoring, an increase in pyroglutamic acid formation and a decrease in C-terminal lysine were observed after thermal treatments. Acidic variant alterations were associated with asparagine deamidation and N-terminal pyroglutamic acid formation. Post-translational modifications were mainly located at the Fc domain, with methionine oxidation and asparagine deamidation as the main modifications occurring at the Fab domain.In conclusion, these results revealed that prolonged thermal treatment under elevated temperatures induces molecular alterations, thereby facilitating the degradation of IgG1. In addition, our findings indicate that both BIO and OR lots exhibit similar degradation profiles when subjected to thermal treatments.

Amyloidosis involves the deposition of misfolded proteins. Even though it is caused by different pathogenic mechanisms, in aggregate, it shares similar features. Here, we tested and confirmed a hypothesis that an amyloid antibody can be engineered by a few mutations to target a different species. Amyloid light chain (AL) and β-amyloid peptide (Aβ) are two therapeutic targets that are implicated in amyloid light chain amyloidosis and Alzheimer's disease, respectively. Though crenezumab, an anti-Aβ antibody, is currently unsuccessful, we chose it as a model to computationally design and prepare crenezumab variants, aiming to discover a novel antibody with high affinity to AL fibrils and to establish a technology platform for repurposing amyloid monoclonal antibodies. We successfully re-engineered crenezumab to bind both Aβ42 oligomers and AL fibrils with high binding affinities. It is capable of reversing Aβ42-oligomers-induced cytotoxicity, decreasing the formation of AL fibrils, and alleviating AL-fibrils-induced cytotoxicity in vitro. Our research demonstrated that an amyloid antibody could be engineered by a few mutations to bind new amyloid sequences, providing an efficient way to reposition a therapeutic antibody to target different amyloid diseases. [ABSTRACT FROM AUTHOR]

Combination antiviral therapy may be helpful in the treatment of SARS-CoV-2 infection; however, no clinical trial data are available, and combined use of direct-acting antivirals (DAA) and monoclonal antibodies (mAb) has been reported only anecdotally. To assess the cooperative effects of dual drug combinations in vitro, we used a VERO E6 cell-based in vitro system with the ancestral B.1 or the highly divergent BQ.1.1 virus to test pairwise combinations of the licensed DAA, including nirmatrelvir (NRM), remdesivir (RDV) and the active metabolite of molnupiravir (EIDD-1931) as well the combination of RDV with four licensed mAbs (sotrovimab, bebtelovimab, cilgavimab, tixagevimab; tested only with the susceptible B.1 virus). According to SynergyFinder 3.0 summary and weighted scores, all the combinations had an additive effect. Within DAA/DAA combinations, paired scores with the B.1 and BQ.1.1 variants were comparable. In the post hoc analysis weighting synergy by concentrations, several cases of highly synergistic scores were detected at specific drug concentrations, both for DAA/DAA and for RDV/mAb combinations. This was supported by in vitro confirmation experiments showing a more than a linear shift of a drug-effective concentration (IC50) at increasing concentrations of the companion drug, although the effect was prominent with DAA/DAA combinations and minimal or null with RDV/mAb combinations. These results support the cooperative effects of dual drug combinations in vitro, which should be further investigated in animal models before introduction into the clinic. [ABSTRACT FROM AUTHOR]

The semi and fully continuous production of monoclonal antibodies (mAbs) has been gaining traction as a lower cost, and efficient production of mAbs to broaden patient access. To be truly flexible and adaptive to process demands, the industry has lacked sufficient advanced control strategies. The variation of the upstream product concentration typically cannot be handled by the downstream capture step, which is configured for a constant feed concentration and fixed binding capacity. This inflexibility leads to losses of efficiency and product yield. This study shows that these challenges can be overcome by a novel advanced control strategy concept that includes dynamic control throughout a perfusion bioreactor, with cell retention by alternating tangential flow, integrated with simulated moving bed (SMB) multi‐column chromatography. The automation workflow and advanced control strategy were implemented through the use of a visual programming development environment. This enabled dynamic flow control across the upstream and downstream process integrated with a dynamic column loading of the SMB. A sensor prototype, based on continuous biolayer interferometry measurements was applied to detect mAb breakthrough within the last column flow‐through to manage column switching. This novel approach provided higher specificity and lower background signal compared to commonly used spectroscopy methods, resulting in an optimized resin utilization while simultaneously avoiding product loss. The dynamic loading was found to provide a twofold increase of the mAb concentration in the eluate compared to a conservative approach with a predefined recipe with similar impurity removal. This concept shows that advanced control strategies can lead to significant process efficiency and yield improvement. [ABSTRACT FROM AUTHOR]

Introduction: Treatment with monoclonal antibodies provides rapid, passive immunity and may stop COVID-19 disease progression. The study evaluated the effect of bamlanivimab (BAM) or BAM + etesevimab (ETE)/sotrovimab compared to placebo on SARS-CoV-2 viral load in patients with COVID-19. Methods: The phase 2, randomized, single-dose study included patients aged between ≥ 18 and < 65 years, not hospitalized at the time of randomization, and had ≥ 1 mild or moderate COVID-19 symptoms. Study included arms 1–6 (placebo, BAM 175 mg + ETE 350 mg, BAM 700 mg + ETE 1400 mg, BAM 2800 mg + ETE 2800 mg, BAM 700 mg alone, and BAM 350 mg + ETE 700 mg, respectively), BAM 700 mg + ETE 700 mg unintentional dosing; and arms 7 and 8 (BAM 700 mg + sotrovimab 500 mg and placebo, respectively). The primary endpoint was proportion of patients with SARS-CoV-2 log viral load > 5.27 on day 7 (persistently high viral load [PHVL]) who received BAM or BAM + (ETE or sotrovimab). Results: A total of 725 patients, mean age 39.6 years (range 18–75 years), 50.2% male were randomized and infused with study drug in arms 1–6; and a total 202 patients, mean age 38 years (range 18–63 years), 53.5% female were randomized and infused with study drug in arms 7 and 8. A significantly lower proportion of patients in arms 2–6 and arm 7 experienced PHVL on day 7 compared to placebo. On day 7, patients in arms 2, 3, and 6 consistently experienced significantly greater reduction in viral load than placebo. Significant improvement was observed in time to viral load clearance and time to symptom improvement by day 29 in some arms compared to placebo. No new safety concerns were observed with drug combinations. Conclusion: The study demonstrated that a significantly lower proportion of patients with mild-to-moderate COVID-19 treated with BAM or BAM + (ETE or sotrovimab) experienced a PHVL at day 7. Trial Registration: ClinicalTrials.gov identifier, NCT04634409. [ABSTRACT FROM AUTHOR]

Developing a preventative vaccine for HIV-1 has been a global priority. The elicitation of broadly neutralizing antibodies (bNAbs) against a broad range of HIV-1 envelopes (Envs) from various strains appears to be a critical requirement for an efficacious HIV-1 vaccine. To understand their ability to neutralize HIV-1, it is important to characterize the binding characteristics of bNAbs. Our work is the first to utilize microscale thermophoresis (MST), a rapid, economical, and flexible in-solution temperature gradient method to quantitatively determine the binding affinities of bNAbs and non-neutralizing monoclonal antibodies (mAbs) to HIV-1 recombinant envelope monomer and trimer proteins of different subtypes, pseudoviruses (PVs), infectious molecular clones (IMCs), and cells expressing the trimer. Our results demonstrate that the binding affinities were subtype-dependent. The bNAbs exhibited a higher affinity to IMCs compared to PVs and recombinant proteins. The bNAbs and mAbs bound with high affinity to native-like gp160 trimers expressed on the surface of CEM cells compared to soluble recombinant proteins. Interesting differences were seen with V2-specific mAbs. Although they recognize linear epitopes, one of the antibodies also bound to the Envs on PVs, IMCs, and a recombinant trimer protein, suggesting that the epitope was not occluded. The identification of epitopes on the envelope surface that can bind to high affinity mAbs could be useful for designing HIV-1 vaccines and for down-selecting vaccine candidates that can induce high affinity antibodies to the HIV-1 envelope in their native conformation. [ABSTRACT FROM AUTHOR]

Introduction: Apicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH&lowbar;00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2). Methods: The cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in Escherichia coli BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in E. necatrix, respectively. Finally, the recombinant pEGFP-C1-DNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein. Results: The EnApiAP2 gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an a-helix and three antiparallel b-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6His tag mAb and the serum of convalescent chickens after infection with E. necatrix, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of EnApiAP2 was significantly higher in SZ The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the DNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein. Discussion: EnApiAP2 protein encoded by ENH&lowbar;00027130 sequence was localized in the nucleus of E. necatrix parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study. [ABSTRACT FROM AUTHOR]

Background: Porcine epidemic diarrhea (PED), caused by PED virus (PEDV), is a severe enteric disease burdening the global swine industry in recent years. Especially, the mortality of PED in neonatal piglets approaches 100%. Maternal antibodies in milk, particularly immunoglobulin A (IgA) antibodies, are of great importance for protection neonatal suckling piglets against PEDV infection as passive lactogenic immunity. Therefore, appropriate detection methods are required for detecting PEDV IgA antibodies in milk. In the current study, we prepared monoclonal antibodies (mAbs) against PEDV spike (S) glycoprotein. An enzyme-linked immunosorbent assay (ELISA) was subsequently developed based on PEDV antigen capture by a specific anti-S mAb. Results: The developed ELISA showed high sensitivity (the maximum dilution of milk samples up to 1:1280) and repeatability (coefficient of variation values < 10%) in detecting PEDV IgA antibody positive and negative milk samples. More importantly, the developed ELISA showed a high coincidence rate with a commercial ELISA kit for PEDV IgA antibody detection in clinical milk samples. Conclusions: The developed ELISA in the current study is applicable for PEDV IgA antibody detection in milk samples, which is beneficial for evaluating vaccination efficacies and neonate immune status against the virus. [ABSTRACT FROM AUTHOR]

Background: Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. Methods: An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. Results: A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M−1 and 2.08 × 107 M−1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. Conclusion: The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics. [ABSTRACT FROM AUTHOR]

Objective: Available guidelines are ambiguous about safe handling monoclonal antibodies (MABs) and whether or not to use a Closed System Drug-Transfer Device (CSTD). In this article we want to describe a standardized working method on handling MABs in a clinical trial setting. Data Sources: The current workflow at the clinical trial unit of the Ghent University Hospital was critically analyzed, after which an extensive literature review was performed using the National Institute for Occupational Safety and Health Working Group guidelines and the database PubMed (Keywords: monoclonal antibodies, closed system transfer devices, safety guidelines, safe handling, management, administration, (bio)compatibility, volume loss, contamination, clinical trial unit. Period: 2020–2022). Data Summary: Literature data are ambiguous. CSTDs can reduce cross-contamination and minimize exposure to potential hazardous drugs for healthcare professionals. However, in recent years more questions have been raised about their in-use compatibility and their impact on final product quality. This makes the debate on implementing CSTDs a hot topic in daily pharmacy practice and demands a holistic and standardized approach when deciding whether or not to use a CSTD when handling MABs. In a clinical trial setting, where safety data are frequently not available and the compatibility of CSTDs and investigational product is often unknown, this poses additional challenges that need to be taken into account. Conclusion: We developed a flowchart which standardizes the use of a CSTD when handling MABs. It allows other healthcare professionals and clinical trial sponsors to define and evaluate the necessary criteria when standardizing the position of a CSTD in their safe handling procedures. [ABSTRACT FROM AUTHOR]