Identification of a linear B-cell epitope on the "puff" loop of the Senecavirus A VP2 protein involved in receptor binding.

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

Tailoring Treatment in Cardiovascular Diseases: The Role of Targeted Therapies.

Cardiovascular diseases (CVDs) remain the leading cause of morbidity and mortality around the globe. To address this public health burden, innovative therapeutic agents are being developed to specifically target molecular and genetic markers. Various therapeutic modalities have been implemented, including vaccines, monoclonal or bispecific antibodies, and gene-based therapies. Such drugs precisely target the underlying disease pathophysiology, aiming at notable molecules such as lipid metabolism regulators, proinflammatory cytokines, and growth factors. This review focuses on the latest advancements in different targeted therapies. It provides an insightful overview of the current landscape of targeted cardiovascular therapies, highlighting promising strategies with potential to transform the treatment of CVDs into an era of precision medicine.

Dupilumab-Related Diabetes Mellitus With Reversal of Symptoms in a Non-genetically Predisposed Patient.

Dupilumab is a fully humanized monoclonal antibody that binds to IL-4 receptors and blocks IL-4 and IL-13 mediated T-helper 2 (Th2) responses. Dupilumab is estimated to be used by over 600,000 patients worldwide for the treatment of atopic dermatitis and other immunologic conditions. Recently, a 66-year-old male patient being treated for atopic dermatitis with dupilumab presented to the clinic with complaints of polyuria and polydipsia. Upon initial testing, the patient was found to have considerable hyperglycemia. Upon genetic testing, he showed no predisposition for autoimmune diabetes and was negative for type I diabetes mellitus-associated human leukocyte antigen (HLA) genes. After immediate cessation of dupilumab, and with subsequent insulin therapy, the patient was able to obtain glycemic control. Following taper and eventual cessation of insulin therapy and over the course of seven months, the patient was able to achieve a full resolution of symptoms and his glycosylated hemoglobin (HgBA1c) levels returned to normal ranges. This case represents only the second documented case of dupilumab-induced diabetes mellitus and is the first known documented case of dupilumab-induced diabetes mellitus in a non-genetically predisposed individual. This case also describes a previously unobserved spontaneous resolution of symptoms upon cessation of the drug. This case further illustrates the potential existence of immunogenic or immunomodulatory side effects of the monoclonal antibody dupilumab that can affect patients who are both genetically and non-genetically predisposed to autoimmune diabetes mellitus.

A low-temperature SPR-based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B.

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.

Preparation and characterization of monoclonal antibodies against porcine gasdermin D protein.

Pyroptosis is a newly discovered type of pro-inflammatory programmed cell death that plays a vital role in various processes such as inflammations, immune responses, and pathogen infections. As one of the main executioners of pyroptosis, gasdermin D (GSDMD) is a membrane pore-forming protein that typically exists in a self-inhibitory state. Once activated, GSDMD will be cleaved into an N-terminal fragment with pore-forming activity, becoming the key indicator of pyroptosis activation, and a C-terminal fragment. Although commercial antibodies against human and murine GSDMD proteins are currently available, their reactivity with porcine GSDMD (pGSDMD) is poor, which limits research on the biological functions of pGSDMD and pyroptosis in pigs in vivo and in vitro. Here, five monoclonal antibodies (mAbs) were prepared by immunizing BALB/c mice with procaryotically expressed full-length pGSDMD, all of which did not cross react with human and murine GSDMD proteins. Epitope mapping demonstrated that 15H6 recognizes amino acids (aa) at positions 28-34 of pGSDMD (LQTSDRF), 19H3 recognizes 257-260aa (PPQF), 23H10 and 27A10 recognize 78-82aa (GPFYF), and 25E2 recognizes 429-435aa (PPTLLGS). The affinity constant and isotype of 15H6, 19H3, 23H10, 27A10, and 25E2 mAbs were determined to be 1.32 × 10-9, 3.66 × 10-9, 9.04 × 10-9, 1.83 × 10-9, and 8.00 × 10-8 mol/L and IgG1/κ, IgG2a/κ, IgG2a/κ, IgG1/κ, and IgG1/κ, respectively. Heavy- and light-chain variable regions sequencing showed that the heavy-chain complementarity-determining region (CDR) sequences of all five mAbs are completely different, while the light-chain CDR sequences of the four mAbs that recognize the N-terminus of pGSDMD are identical. Our prepared mAbs provide valuable materials for studying pGSDMD function and pyroptosis. KEY POINTS: • A total of five mouse anti-pGSDMD mAbs were prepared, of which four recognize the N-terminus of pGSDMD and one recognize its C-terminus. • The main performance parameters of the five mAbs, including epitope, antibody titer, affinity constant, isotype, and heavy- and light-chain CDR, were characterized. • All five mAbs specifically recognize pGSDMD protein and do not cross react with human and murine GSDMD proteins.

Immunological Quantitation of the Glycation Site Lysine-414 in Serum Albumin in Human Plasma Samples by Indirect ELISA Using Highly Specific Monoclonal Antibodies.

Diabetes mellitus, a metabolic disorder that is characterized by elevated blood glucose levels, is common throughout the world and its prevalence is steadily increasing. Early diagnosis and treatment are important to prevent acute complications and life-threatening long-term organ damage. Glycation sites in human serum albumin (HSA) are considered to be promising biomarkers of systemic glycemic status. This work aimed to develop a sensitive and clinically applicable ELISA for the quantification of glycation site Lys414 in HSA (HSAK414 ). The monoclonal antibodies (mAbs) were generated by immunizing mice with a glycated peptide. The established indirect ELISA based on mAb 50D8 (IgG1 isotype) yielded a limit of detection of 0.39 nmol/g HSA for HSAK414 with a linear dynamic range from 0.50 to 6.25 nmol/g glycated HSA. The inter- and intra-day assays with coefficients of variation less than 20 % indicated good assay performance and precision. Assay evaluation was based on plasma samples from diabetic and non-diabetic subjects with known HSAK414 glycation levels previously determined by LC-MS. Both data sets correlated very well. In conclusion, the generated mAb 50D8 and the established ELISA could be a valuable tool for the rapid quantitation of glycation site HSAK414 in plasma samples to evaluate its clinical relevance.

Benchmark Glycan Profile of Therapeutic Monoclonal Antibodies Produced by Mammalian Cell Expression Systems.

PURPOSE: This study aims to establish a benchmark glycan profile for commercial therapeutic monoclonal antibodies (mAbs) approved by the US Food and Drug Administration (FDA). METHODS: We conducted a rigorous comparison of glycosylation data from the regulatory submissions for FDA-approved therapeutic antibodies up to May 2023. This analysis includes over 150 mAbs produced by various mammalian cell expression systems. RESULTS: The study identified nine prevalent glycan epitopes across all FDA-approved monoclonal antibodies produced by different expression systems. These epitopes include terminal N-acetylglucosamine, core fucose, terminal galactose, high mannose, α-galactose, terminal α2,3-linked N-acetylneuraminic acid, terminal α2,6-linked N-glycolylneuraminic acid, triantennary structure, and bisecting N-acetylglucosamine, thus establishing a benchmark glycan profile. CONCLUSIONS: The findings of this study have significant implications for therapeutic antibody development, quality control, and regulatory compliance. The benchmark glycan profile enables the assessment of glycosylation consistency and comparability across a diverse range of antibody products, ensuring improved product quality within the biopharmaceutical industry.

Alleviation of the necessity for supernatant prefiltering in the protein a recovery of Monoclonal antibodies from Chinese hamster ovary cell cultures.

Protein A (ProA) chromatography is a mainstay in the analytical and preparative scale isolation/purification of monoclonal antibodies (mAbs). One area of interest is continuous processing or continuous chromatography, where ProA chromatography is used in the large-scale purification of mAbs. However, filtration is required prior to all ProA isolations to remove large particulates in cell culture supernatant, consisting of a mixture of cell debris, host cell contaminants, media components, etc. Currently, in-line filters are used to remove particles in the supernatant, requiring replacement over time due to fouling; regardless of the scale. Here we demonstrate the ProA isolation of unfiltered Chinese hamster ovary (CHO) cell media using capillary-channel polymer (C-CP) fiber stationary phases modified with S. aureus Protein A (rSPA). The base polymer of the analytical scale C-CP columns costs ∼$5 per 30 cm column, and when modified with ProA, the base cost is ∼$25 per 30 cm column, a cost-effective option in comparison to analytical-scale commercial columns. To directly sample unfiltered media, a 5 cm gap was created at the head of the C-CP column, where the large particulates are trapped, while molecular solutes flow through the capillary channels without sacrifice in analytical performance, mAb loading capacity, or backpressure increases. The binding capacity of the gap ProA C-CP column was âˆ¼ 2 mg mL-1 of IgG per bed volume. The same analytical column could be operated after processing a total of âˆ¼ 56 column bed volumes of supernatant (>25 analytical cycles) without the need for caustic clean-in-place processing.

Treatment Patterns of Monoclonal Antibodies in Patients With Severe Uncontrolled Asthma Treated by Pulmonologists in Spain.

Introduction and objectives: The use of monoclonal antibody (mAb)-based therapies is becoming the new standard of care for severe uncontrolled asthma (SUA). Even though patients may qualify for one or more of these targeted treatments, based on different clinical criteria, a global vision of mAb prescription management in a large sample of hospitals is not well characterised in Spain.The objective was to give a global vision of mAb prescription management in a large sample of hospitals in Spain. Materials and methods: We used an aggregate data survey method to interview pulmonology specialists in a large sample of Spanish centres (90). The following treatment-related information was obtained on patients treated with mAbs: specific mAbs prescribed, treatment interruption, switch and restart and the reasons for these treatment changes. Results: mAb prescription was more frequent in females (13.3% females vs 7.4% males; p < 0.001). There were no differences in prevalence by hospital complexity level. In contrast, there were differences by geographical area. Omalizumab was the most prescribed mAb (6.2%), followed by mepolizumab (2.9%). Discontinuation of Omalizumab (due to a lack of effectivity) and switches from this mAb to mepolizumab were more frequent. Very few restarts to the first treatment were observed after a switch from ≥2 mAbs. Conclusions: Omalizumab appeared as the most prescribed mAb in SUA but was also the most withdrawn; a specific and objective characterisation of patients with SUA, along with asthma phenotyping, and together with further evaluation of safety and effectiveness profiles, will lead to future progress in the management of SUA with mAbs.

Introducción y objetivos: El uso de terapias basadas en anticuerpos monoclonales (mAb) se está convirtiendo en el nuevo estándar de atención para el asma grave no controlada (AGNC). A pesar de que los pacientes pueden optar a uno o varios de estos tratamientos dirigidos, con base en diferentes criterios clínicos, en España no se ha caracterizado bien una visión global de la gestión de la prescripción de mAb en una gran muestra de hospitales.El objetivo fue dar una visión global de la gestión de la prescripción de mAB en una amplia muestra de hospitales en España. Materiales y métodos: Se utilizó un método basado en una encuesta de datos agregados para entrevistar a especialistas en Neumología en una amplia muestra de centros españoles (90). Se obtuvo la siguiente información relacionada con el tratamiento de los casos tratados con mAbs: mAbs específicos prescritos, interrupción del tratamiento, cambio y reinicio, y las razones de estos cambios de tratamiento en consultas de Neumología. Resultados: La prescripción de mAB fue más frecuente en mujeres (13,3% mujeres vs. 7,4% hombres; p < 0,001). No hubo diferencias de prevalencia por nivel hospitalario. En cambio, hubo diferencias por área geográfica. Omalizumab fue el mAb más prescrito (6,2%), seguido de mepolizumab (2,9%). La interrupción y los cambios (debido a la falta de efectividad) también fueron más frecuentes para omalizumab. Conclusiones: Omalizumab fue el mAb más prescrito en el manejo de AGNC, pero también fue el mAB que presentó más retiradas; una caracterización específica y objetiva de los pacientes con AGNC, mediante fenotipificación de asma, junto con una evaluación adicional de los perfiles de seguridad y efectividad, conducirá a nuevos avances en el manejo del AGNC con mABs.

Tixagevimab/Cilgavimab as pre-exposure prophylaxis against SARS-CoV-2 in patients with hematological malignancies.

The approved combination of Tixagevimab/Cilgavimab has been shown to decrease the rate of symptomatic SARS-CoV-2 infection in patients at increased risk of inadequate response to vaccination. However, Tixagevimab/Cilgavimab was tested in a few studies that included patients with hematological malignancies, even if this population has shown an increased risk of unfavorable outcomes following infection (with high rates of hospitalization, intensive care unit admission, and mortality) and poor significant immunization following vaccines. We performed a real-life prospective cohort study to evaluate the rate of SARS-CoV-2 infection following pre-exposure prophylaxis with Tixagevimab/Cilgavimab in anti-spike seronegative patients compared to a cohort of seropositive patients who were observed or received a fourth vaccine dose. We recruited 103 patients with a mean age of 67 years: 35 (34%) received Tixagevimab/Cilgavimab and were followed from March 17, 2022, until November 15, 2022. After a median follow-up of 4.24 months, the 3-month cumulative incidence of infection was 20% versus 12% in the Tixagevimab/Cilgavimab and observation/vaccine groups respectively (HR 1.57; 95% CI: 0.65-3.56; p = 0.34). In this study, we report our experience with Tixagevimab/Cilgavimab and a tailored approach to SARS-CoV-2 infection prevention in patients with hematological malignancies during the SARS-CoV-2 omicron surge.

Fragmented antibodies in non-small cell lung cancer: A novel nano-engineered delivery system for detection and treatment of cancer.

Non-small cell lung cancer (NSCLC) has a long history of defying traditional cytotoxic treatment. Significant advancements in biotechnology, cancer biology, and immunotherapy have provided new insights that have altered the landscape for the management of NSCLC, clearing the way for a new era of pharmaceuticals in the form of monoclonal antibodies and their fragments. Antibody fragments are superior to monoclonal antibodies because of their small size, which allows them to penetrate cells and tissues effectively. When combined with functional nanocarriers, antibody fragments can target cancer cells while offering improved efficacy and fewer off-target effects. We discuss current topics of interest including anti-CTLA-4 mAbs, Talactoferrin alfa (TLF), and the CYFRA 21-1 biomarker, with brief insights into its novel detection system.

Novel targeted inhibition of the IL-5 axis for drug reaction with eosinophilia and systemic symptoms syndrome.

Background: The drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome represents a severe hypersensitivity reaction. Up-to-date treatment is based on withdrawal of medication, supportive care, and immunosuppression using high-dose corticosteroid (CS) therapy. However, evidence-based data are lacking regarding second-line therapy for steroid-resistant or steroid-dependent patients. Objectives: We hypothesize that the interleukin (IL)-5 axis plays a critical role in the pathophysiology of DRESS; hence, inhibition of this signaling pathway could offer a potential therapy for steroid-dependent and/or steroid-resistant cases, and it may offer an alternative to CS therapy in certain patients more prone to CS toxicity. Methods: Herein, we collected worldwide data on DRESS cases treated with biological agents targeting the IL-5 axis. We reviewed all cases indexed in PubMed up to October 2022 and performed a total analysis including our center experience with two additional novel cases. Results: A review of the literature yielded 14 patients with DRESS who were treated with biological agents targeting the IL-5 axis as well as our two new cases. Reported patients are characterized by a female-to-male ratio of 1:1 and a mean age of 51.8 (17-87) years. The DRESS-inducing drugs, as expected from the prospective RegiSCAR study, were mostly antibiotics (7/16), as follows: vancomycin, trimethoprim-sulfamethoxazole, ciprofloxacin, piperacillin-tazobactam, and cefepime. DRESS patients were treated with anti-IL-5 agents (mepolizumab and reslizumab) or anti-IL-5 receptor (IL-5R) biologics (benralizumab). All patients have clinically improved under anti-IL-5/IL-5R biologics. Multiple doses of mepolizumab were needed to achieve clinical resolution, whereas a single dose of benralizumab was often sufficient. Relapse was noted in one patient receiving benralizumab treatment. One patient receiving benralizumab had a fatal outcome, although mortality was probably related to massive bleeding and cardiac arrest due to coronavirus disease 2019 (COVID-19) infection. Conclusion: Current treatment guidelines for DRESS are based on case reports and expert opinion. Understanding the central role of eosinophils in DRESS pathogenicity emphasizes the need for future implementation of IL-5 axis blockade as steroid-sparing agents, potential therapy to steroid-resistant cases, and perhaps an alternative to CS treatment in certain DRESS patients more prone to CS toxicity.

Efficacy and safety of monoclonal antibody therapy in patients with neuromyelitis optica spectrum disorder: A systematic review and network meta-analysis.

Introduction: Neuromyelitis optica spectrum disorder (NMOSD) is a devastating inflammatory CNS demyelinating disease. Two groups of monoclonal antibodies (mAbs) are used to prevent disease relapse, i.e., Food and Drug Administration (FDA)-approved mAbs (e.g., eculizumab satralizumab, inebilizumab), and off-label mAb drugs (e.g., rituximab and tocilizumab). The FDA-approved mAbs have high efficacy but more expensive compared to the off-labels, and thus are less accessible. This systematic review and network meta-analysis (NMA) was to assess the efficacy and safety of both classes of mAbs compared to the current standard treatments. Methods: Systematically searches were conducted in MEDLINE and SCOPUS from inception until July 2021. Randomized-controlled trials (RCTs) were eligible if they compared any pair of treatments (mAbs, immunosuppressive drugs, or placebo) in adult patients with NMOSD. Studies with AQP4-IgG positive or negative were used in the analysis. Probability of relapse and time to event were extracted from the Kaplan-Meier curves using Digitizer. These data were then converted into individual patient time-to-event data. A one-stage mixed-effect survival model was applied to estimate the median time to relapse and relative treatment effects using hazard ratios (HR). Two-stage NMA was used to determine post-treatment annualized relapse rate (ARR), expanded disability status score (EDSS) change, and serious adverse events (SAE). Risk of bias was assessed using the revised cochrane risk of bias tool. Results: A total of 7 RCTs with 776 patients were eligible in the NMA. Five of the seven studies were rated low risk of bias. Both FDA-approved and off-label mAbs showed significantly lower risk of relapse than standard treatments, with HR (95% CI) of 0.13 (0.07, 0.24) and 0.16 (0.07, 0.37) respectively. In addition, the FDA-approved mAbs had 20% lower risk of relapse than the off-label mAbs, but this did not reach statistical significance. The ARRs were also lower in FDA-approved and off-label mAbs than the standard treatments with the mean-difference of-0.27 (-0.37,-0.16) and-0.31(-0.46,-0.16), respectively. Conclusion: The off-label mAbs may be used as the first-line treatment for improving clinical outcomes including disease relapse, ARR, and SAEs for NMOSD in countries where resources and accessibility of the FDA-approved mAbs are limited. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=283424, identifier: CRD42021283424.

Enhancing pharmacokinetic and pharmacodynamic properties of recombinant therapeutic proteins by manipulation of sialic acid content.

The circulatory half-life of recombinant therapeutic proteins is an important pharmacokinetic attribute because it determines the dosing frequency of these drugs, translating directly to treatment cost. Thus, recombinant therapeutic glycoproteins such as monoclonal antibodies have been chemically modified by various means to enhance their circulatory half-life. One approach is to manipulate the N-glycan composition of these agents. Among the many glycan constituents, sialic acid (specifically, N-acetylneuraminic acid) plays a critical role in extending circulatory half-life by masking the terminal galactose that would otherwise be recognised by the hepatic asialoglycoprotein receptor (ASGPR), resulting in clearance of the biotherapeutic from the circulation. This review aims to provide an illustrative overview of various strategies to enhance the pharmacokinetic/pharmacodynamic properties of recombinant therapeutic proteins through manipulation of their sialic acid content.

Leading Edge: Intratumor Delivery of Monoclonal Antibodies for the Treatment of Solid Tumors.

Immunotherapies based on immune checkpoint blockade have shown remarkable clinical outcomes and durable responses in patients with many tumor types. Nevertheless, these therapies lack efficacy in most cancer patients, even causing severe adverse events in a small subset of patients, such as inflammatory disorders and hyper-progressive disease. To diminish the risk of developing serious toxicities, intratumor delivery of monoclonal antibodies could be a solution. Encouraging results have been shown in both preclinical and clinical studies. Thus, intratumor immunotherapy as a new strategy may retain efficacy while increasing safety. This approach is still an exploratory frontier in cancer research and opens up new possibilities for next-generation personalized medicine. Local intratumor delivery can be achieved through many means, but an attractive approach is the use of gene therapy vectors expressing mAbs inside the tumor mass. Here, we summarize basic, translational, and clinical results of intratumor mAb delivery, together with descriptions of non-viral and viral strategies for mAb delivery in preclinical and clinical development. Currently, this is an expanding research subject that will surely play a key role in the future of oncology.

Preparation and characterization of a neutralizing murine monoclonal antibody against tetanus toxin.

After Clostridium tetani infects the human body, it propagates under anaerobic conditions and produces tetanus neurotoxin (TeNT). TeNT can affect the central nervous system, inhibit the release of neurotransmitters, and result in respiratory failure, which are the root causes of death in tetanus patients. Identifying monoclonal antibodies (mAbs) targeting TeNT with neutralizing activity is urgently needed for the prevention and treatment of tetanus infection. In this study, through immunizing BALB/c mice with tetanus toxoid (TT), we obtained six positive hybridoma cell lines (1A7, 2C7, 3A7, 3H4, 4C1, and 4E12). Antibody isotyping showed that the antibodies are all of the IgG1/κ subclass. Ascites fluid was prepared by allogeneic ascites induction and the antibodies were purified through protein G affinity chromatography columns. Purities of the produced murine mAbs were all greater than 95%. All six antibodies bound to linear epitopes, among which 3A7 bound to the TeNT/L domain and the other five antibodies bound to the TeNT/Hc domain. Moreover, the affinity constants of these six antibodies against the antigen were all in the nanomolar range, and the affinity of 4E12 antibody reached the picomolar range. Results from toxin-neutralization assays in mice showed that 2C7 antibody delayed animal death, while 1A7, 3A7, 3H4, and 4E12 antibodies conferred partial protection. Additionally, 4C1 antibody offered complete protection, as 200 µg of 4C1 antibody fully protected against toxin challenge with 10 LD50 of TeNT and had a window period of 1 h. Antibody epitope grouping results revealed that the binding epitopes of 4C1 antibody were different from those of the other five antibodies. When 4C1 antibody was used in combination with another antibody, the neutralizing activities of antibodies were all evidently enhanced. Specifically, 4C1 combined with 3A7 antibody led to the greatest improvement in neutralizing activities, and 20 µg antibodies total (10 + 10 µg) fully protected against toxin challenge with 10 LD50. When 4E12, 3A7, and 4C1 antibodies were used in combination, 18 µg antibodies total (6 + 6 + 6 µg) completely neutralized 10 LD50 toxin. The present study derived murine mAbs with neutralizing activities and laid the foundation for follow-up therapeutic drug development for TeNT poisoning as well as establishment of TeNT detection methods.

Microporous affinity membranes and their incorporation into microfluidic devices for monitoring of therapeutic antibodies.

Control of monoclonal antibody (mAb) concentrations in serum is important for maintaining the safety and efficacy of these lifesaving therapeutics. Point-of-care (POC) quantification of therapeutic mAbs could ensure that patients have effective mAb levels without compromising safety. This work uses mimotope-functionalized microporous alumina affinity membranes in vertical flow assays for detection and quantitation of therapeutic mAbs. Selective capture of bevacizumab from 1000:1 diluted serum or plasma and binding of a fluorescently labelled anti-human IgG secondary antibody enable fluorescence-based analysis of bevacizumab at its therapeutically relevant concentration range of ∼50-300 µg/mL. The assay results in a linear relationship between the fluorescence intensity of the antibody capture spot and the bevacizumab concentration. A simple prototype microfluidic device containing these membranes allows washing, reagent additions and visualization of signal within 15 min using a total of 5 mL of fluid. The prototype devices can monitor physiologically relevant bevacizumab levels in diluted serum, and future refinements might lead to a POC device for therapeutic drug monitoring.

Antibody-Based Immunotherapies as a Tool for Tackling Multidrug-Resistant Bacterial Infections.

The discovery of antimicrobials is an outstanding achievement of mankind that led to the development of modern medicine. However, increasing antimicrobial resistance observed worldwide is rendering commercially available antimicrobials ineffective. This problem results from the bacterial ability to adapt to selective pressure, leading to the development or acquisition of multiple types of resistance mechanisms that can severely affect the efficacy of antimicrobials. The misuse, over-prescription, and poor treatment adherence by patients are factors strongly aggravating this issue, with an epidemic of infections untreatable by first-line therapies occurring over decades. Alternatives are required to tackle this problem, and immunotherapies are emerging as pathogen-specific and nonresistance-generating alternatives to antimicrobials. In this work, four types of antibody formats and their potential for the development of antibody-based immunotherapies against bacteria are discussed. These antibody isotypes include conventional mammalian polyclonal antibodies that are used for the neutralization of toxins; conventional mammalian monoclonal antibodies that currently have 100 IgG mAbs approved for therapeutic use; immunoglobulin Y found in birds and an excellent source of high-quality polyclonal antibodies able to be purified noninvasively from egg yolks; and single domain antibodies (also known as nanobodies), a recently discovered antibody format (found in camelids and nurse sharks) that allows for a low-cost synthesis in microbial systems, access to hidden or hard-to-reach epitopes, and exhibits a high modularity for the development of complex structures.

Production of two SARS-CoV-2 neutralizing antibodies with different potencies in Nicotiana benthamiana.

Monoclonal antibodies are considered to be highly effective therapeutic tools for the treatment of mild to moderate COVID-19 patients. In the present work, we describe the production of two SARS-CoV-2 human IgG1 monoclonal antibodies recognizing the spike protein receptor-binding domain (RBD) and endowed with neutralizing activity (nAbs) in plants. The first one, mAbJ08-MUT, was previously isolated from a COVID-19 convalescent patient and Fc-engineered to prolong the half-life and reduce the risk of antibody-dependent enhancement. This nAb produced in mammalian cells, delivered in a single intramuscular administration during a Phase I clinical study, was shown to (i) be safe and effectively protect against major variants of concern, and (ii) have some neutralizing activity against the recently emerged omicron variant in a cytopathic-effect-based microneutralization assay (100% inhibitory concentration, IC100 of 15 µg/mL). The second antibody, mAb675, previously isolated from a vaccinated individual, showed an intermediate neutralization activity against SARS-CoV-2 variants. Different accumulation levels of mAbJ08-MUT and mAb675 were observed after transient agroinfiltration in Nicotiana benthamiana plants knocked-out for xylosil and fucosil transferases, leading to yields of ~35 and 150 mg/kg of fresh leaf mass, respectively. After purification, as a result of the proteolytic events affecting the hinge-CH2 region, a higher degradation of mAb675 was observed, compared to mAbJ08-MUT (~18% vs. ~1%, respectively). Both nAbs showed a human-like glycosylation profile, and were able to specifically bind to RBD and compete with angiotensin-converting enzyme 2 binding in vitro. SARS-CoV-2 neutralization assay against the original virus isolated in Wuhan demonstrated the high neutralization potency of the plant-produced mAbJ08-MUT, with levels (IC100 < 17 ng/mL) comparable to those of the cognate antibody produced in a Chinese hamster ovary cell line; conversely, mAb675 exhibited a medium neutralization potency (IC100 ~ 200 ng/mL). All these data confirm that plant expression platforms may represent a convenient and rapid production system of potent nAbs to be used both in therapy and diagnostics in pandemic emergencies.

A New Alternative Tool to Analyse Glycosylation in Monoclonal Antibodies Based on Drop-Coating Deposition Raman imaging: A Proof of Concept.

Glycosylation is considered a critical quality attribute of therapeutic proteins as it affects their stability, bioactivity, and safety. Hence, the development of analytical methods able to characterize the composition and structure of glycoproteins is crucial. Existing methods are time consuming, expensive, and require significant sample preparation, which can alter the robustness of the analyses. In this context, we developed a fast, direct, and simple drop-coating deposition Raman imaging (DCDR) method combined with multivariate curve resolution alternating least square (MCR-ALS) to analyze glycosylation in monoclonal antibodies (mAbs). A database of hyperspectral Raman imaging data of glycoproteins was built, and the glycoproteins were characterized by LC-FLR-MS as a reference method to determine the composition in glycans and monosaccharides. The DCDR method was used and allowed the separation of excipient and protein by forming a "coffee ring". MCR-ALS analysis was performed to visualize the distribution of the compounds in the drop and to extract the pure spectral components. Further, the strategy of SVD-truncation was used to select the number of components to resolve by MCR-ALS. Raman spectra were processed by support vector regression (SVR). SVR models showed good predictive performance in terms of RMSECV, R2CV.