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The site-specific antibody–drug conjugates (ADCs), particularly those utilizing the engineered cysteine in Fc fragments of mAbs (THIOMAB™ antibodies), have emerged as a novel class of biotherapeutics for cancer treatment. The engineered cysteine residues in these antibodies are capped by cysteine or glutathione through a disulfide bond. Prior to conjugation with linker-payloads, these caps need to be removed through a reduction process. However, monitoring the efficiency of the decapping process has been challenging due to the lack of effective analytical methods. Intact reversed-phase liquid chromatography-mass spectrometry and hydrophobic interaction chromatography methods failed to separate decapped and capped intact THIOMAB™ mAbs in our study. Instead the fragmentation of mAbs provided a novel strategy to analyze the decapping effiency. After cleavage using a hinge specific enzyme, the generated Fc fragments with and without cysteine and/or glutathione caps displayed different hydrophobicity and were well separated by RPLC, allowing quantitative determination of the decapping efficiency. Enzymes that cleave both above and below the hinge disulfide bonds were tested. The use of FabALATICA can determine percentages of molecules with 0, 1, and 2 cysteine and/or glutathione caps, respectively, regardless of whether the antibody contains the hinge LALA mutations. On the other hand, FabRICATOR enzyme can only be utilized for antibodies without LALA mutations for the overall decapping percentage and cannot be used to estimate intact antibody each with 0, 1, and 2 caps. Therefore, FabALACTICA cleavage followed by RPLC provides a wider application of monitoring the decapping efficiency of all antibodies with the engineered cysteine in Fc. [ABSTRACT FROM AUTHOR]

Background: Ensuring regulatory-compliant manufacturing capability is an essential challenge for new treatment modalities, but its internalisation is not easy for pharmaceutical companies, especially start-ups. This study examines the functions and requirements of contracted development and manufacturing organisations (CDMOs) using the development process of antibody medicines as a case study. Methods: Utilizing PubMed, Cortellis and Patent Integration databases, this study delves into publication and contractual trends in monoclonal antibody drugs (mAbs) development, alongside an analysis of patent filings by CDMOs, offering a comprehensive overview of the evolving landscape in mAbs innovation. Results: In the early stages of mAbs development, dedicated bio firms (DBFs) led R&D with superior drug discovery technology but lacked manufacturing capability, which was complemented by CDMOs. This collaboration was an opportunity for CDMOs to expand their capabilities beyond manufacturing technology into antibody drug candidate discovery and structural optimisation technology. From mid-development onwards, it established a technology platform based on these capabilities and developed and established partnerships with existing pharmaceutical companies, including mega pharma. Conclusions: The impact of institutions and regulations on the innovation process was assessed during this development process. These findings are expected to provide valuable insights into the innovation system for new modalities. [ABSTRACT FROM AUTHOR]

Sodium butyrate (NaBu), well-known as a histone deacetylase inhibitor and for its capacity to impede cell growth, can enhance the production of a specific protein, such as an antibody, in recombinant Chinese hamster ovary (CHO) cell cultures. In this study, two CHO cell lines, namely K1 and DG44, along with their corresponding mAb-producing lines, K1-Pr and DG44-Pr, were cultivated with or without NaBu. A SWATH-based profiling method was employed to analyze the proteome. Cells cultured in the presence of NaBu exhibited a reduction in mitosis and gene expression, supported by their culture data demonstrating growth inhibition. The presence of NaBu corresponded to upregulation of intracellular trafficking and secretion pathways, aligned with an observed increase in mAb production, and was associated with an elevated glycosylation pathway and a slight alteration in the glycosylation profile of the mAbs. Increased fatty acid oxidation, redox interactions, and lipid biosynthesis were also observed and are likely attributable to the metabolism of NaBu. A comprehensive understanding of the systemic effects of NaBu will facilitate the discovery of strategies to enhance or prolong the productivity of CHO cells. [ABSTRACT FROM AUTHOR]

Purpose: Pregnant and postpartum women are at increased risk of developing severe COVID-19. Monoclonal antibodies (mAbs) are now widely used in high-income countries to treat mild to moderate COVID-19 outpatients at risk for developing severe disease. Very few data are available on the use of mAbs in special populations, including pregnant and postpartum women. Here we present our early experience with mAbs in these two populations. Methods: Electronic records of pregnant and postpartum women treated with mAbs at Careggi University Hospital, Florence, were retrieved. Relevant data were extracted (age, presence of risk factors for COVID-19, oxygen support, mAb type, gestational age, and pregnancy status). When available, outcomes at 28 days after administration were also included. Results: From March 1st to September 30th 2021, eight pregnant and two postpartum women have been treated with mAbs at our center. The median age was 31 years (IQR 30–33.5, range 29–38), median gestational age was 24 weeks. Seven patients had additional risk factors. According to the Italian disposition, all patients received casirivimab/imdevimab, with five receiving a 2.4 mg dose and five receiving a 8 g dose. Eight patients improved. One developed myocarditis, considered a COVID-19 complication. Another required a transient increase of low flow oxygen support before improving and being discharged. At a 28 days follow-up, all patients were clinically recovered. We did not observe mAbs related adverse events. Conclusion: Although preliminary data should be interpreted with caution, it is remarkable how mAbs were well tolerated by pregnant women with COVID-19. Further data on mAbs in this special population should be collected but the use of mAbs in pregnant and postpartum patients should be considered. Even thus oral antivirals are becoming available, they are not recommended in pregnant and postpartum women. This population may specifically benefit from treatment with last generation mAbs. [ABSTRACT FROM AUTHOR]

SARS-CoV-2, the newly emerged virus of the Coronaviridae family is causing havoc worldwide. The novel coronavirus 2019 was first reported in Wuhan, China marked as the third highly infectious pathogenic virus of the twenty-first century. The typical manifestations of COVID-19 include cough, sore throat, fever, fatigue, loss of sense of taste and difficulties in breathing. Large numbers of SARS-CoV-2 infected patients have mild to moderate symptoms, however severe and life-threatening cases occur in about 5–10% of infections with an approximately 2% mortality rate. For the treatment of SARS-CoV-2, the use of neutralizing monoclonal antibodies (mAbs) could be one approach. The receptor binding domain (RBD) and N-terminal domain (NTD) situated on the peak of the spike protein (S-Protein) of SARS-CoV-2 are immunogenic in nature, therefore, can be targeted by neutralizing monoclonal antibodies. Several bioinformatics approaches highlight the identification of novel SARS-CoV-2 epitopes which can be targeted for the development of COVID-19 therapeutics. Here we present a summary of neutralizing mAbs isolated from COVID-19 infected patients which are anticipated to be a better therapeutic alternative against SARS-CoV-2. However, provided the vast escalation of the disease worldwide affecting people from all strata, affording expensive mAb therapy will not be feasible. Hence other strategies are also being employed to find suitable vaccine candidates and antivirals against SARS-CoV-2 that can be made easily available to the population. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) play an important role in activating, regulating, and maintaining the immune response. CD103+ DCs, one of the DC subpopulations, mainly function in the mucosal immune response. They are responsible for capturing and carrying antigens to the relevant lymph nodes to activate the downstream immune responses. However, there is limited available information regarding the function of CD103+ DCs in the porcine mucosal immune response. In this study, two monoclonal antibodies (mAbs) against porcine CD103 were prepared, and their applications were evaluated by enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence assay (IFA), and flow cytometry. The produced mAbs (7F3 and 9H3) were both IgG1 subtype with κ chains in the light chain. The 7F3 recognizes a linear epitope (PDLRPRAQVYFSDLE) while 9H3 recognizes another linear epitope (QILDEGQVLLGAVGA). The prepared mAbs could be used in vivo to detect the cells expressing CD103 molecules, giving wide applications of both mAbs. In conclusion, this study successfully prepared 2 mAbs against CD103 protein, and they showed applicability in vivo experiments, which will provide the basis for the study of porcine mucosal immunity. Key points: • Preparation of monoclonal antibodies against porcine CD103 molecule • Analysis of the distribution of CD103 protein on different cells is possible • Exploration of the CD103+ DCs function in porcine mucosal immunity is possible [ABSTRACT FROM AUTHOR]

Abstract Some antibacterial therapies entail sequential treatments with different antibiotics, but whether this approach is optimal for anti‐cancer tyrosine kinase inhibitors (TKIs) remains open. EGFR mutations identify lung cancer patients who can derive benefit from TKIs, but most patients develop resistance to the first‐, second‐, and third‐generation drugs. To explore alternatives to such whack‐a‐mole strategies, we simulated in patient‐derived xenograft models the situation of patients receiving first‐line TKIs. Monotherapies comprising approved first‐line TKIs were compared to combinations with antibodies specific to EGFR and HER2. We observed uniform and strong superiority of all drug combinations over the respective monotherapies. Prolonged treatments, high TKI dose, and specificity were essential for drug–drug cooperation. Blocking pathways essential for mitosis (e.g., FOXM1), along with downregulation of resistance‐conferring receptors (e.g., AXL), might underlie drug cooperation. Thus, upfront treatments using combinations of TKIs and antibodies can prevent emergence of resistance and hence might replace the widely applied sequential treatments utilizing next‐generation TKIs.

Purpose: The viscosity of highly concentrated therapeutic monoclonal antibody (mAb) formulations at concentrations ≥ 100 mg/mL can significantly affect the stability, processing, and drug product development for subcutaneous delivery. An early identification of a viscosity prone mAb during candidate selection stages are often beneficial for downstream processes. Higher order structure of mAbs may often dictate their viscosity behavior at high concentration. Thus it is beneficial to gauge or rank-order their viscosity behavior using noninvasive structural fingerprinting methods and to potentially screen for suitable viscosity lowering excipients. Methods: In this study, Dynamic Light Scattering (DLS) and 2D NMR based methyl fingerprinting were used to correlate viscosity behavior of a set of Pfizer mAbs. The viscosities of mAbs were determined. Respective Fab and Fc domains were generated for studies. Result: Methyl fingerprinting of intact mAbs allows for differentiation of viscosity prone mAbs from well behaved ones even at 30–40 mg/ml, where bulk viscosity of the solutions are near identical. For viscosity prone mAbs, peak broadening and or distinct chemical shift changes were noted in intact and fragment fingerprints, unlike the well-behaved mAbs, indicative of protein protein interactions (PPI). Conclusion: Fab-Fab or Fab-Fc interactions may lead to formation of protein networks at high concentration. The early transients to these network formation may be manifested through peak broadening or peak shift in the 2D NMR spectrum of mAb/mAb fragments. Such insights go beyond rank ordering mAbs based on viscosity behavior, which can be obtained by other methods as well.. [ABSTRACT FROM AUTHOR]

Keywords: COVID-19; mAbs; Monoclonal antibodies; SARS-CoV-2; Casirivimab; Imdevimab; Guidelines EN COVID-19 mAbs Monoclonal antibodies SARS-CoV-2 Casirivimab Imdevimab Guidelines 635 638 4 02/16/22 20220201 NES 220201 Dear Editor, We thank Manciulli and colleagues for their comment on the recently released guidelines from the Italian Society of Anti-Infective Therapy (SITA) and the Italian Society of Pulmonology (SIP) on the clinical management of adult patients with coronavirus disease 2019 (COVID-19) outside intensive care units [[1]]. Clinical management of adult patients with COVID-19 outside intensive care units: guidelines from the Italian Society of Anti-Infective Therapy (SITA) and the Italian Society of Pulmonology (SIP). [Extracted from the article]

Purpose of Review: The treatment landscape for multiple myeloma has evolved rapidly with the availability of multiple new drugs; however, although patient survival has improved, the disease remains incurable. Multiple myeloma is characterized by the unregulated growth of malignant plasma cells accompanied by immune dysfunction as well as disrupted immune surveillance mechanisms. Here, we analyze clinical modalities, with a focus on monoclonal antibodies and adoptive cellular therapy that enhance patients' immune systems and overcome these defects.Recent Findings: Early clinical trials with PD-1 inhibitors were promising, but randomized phase III trials with immunomodulatory drugs showed increased toxicities. Monoclonal antibodies targeting surface antigens led to substantial clinical efficiency in relapsed myeloma. Chimeric antigen receptor (CAR) T cell therapy for multiple myeloma represents a significant advance, as exciting and dramatic responses in early clinical trials have been seen.Summary: Immunotherapeutic approaches are promising and can augment or replace the current standard of care, with the potential to offer extended survival for myeloma patients. [ABSTRACT FROM AUTHOR]

We examined whether abnormal volumes of several brain regions as well as their mutual associations that have been observed in patients with schizophrenia, are also present in individuals at clinical high-risk (CHR) for developing psychosis. 3T magnetic resonance imaging was acquired in 19 CHR and 20 age- and handedness-matched controls. Volumes were measured for the body and temporal horns of the lateral ventricles, hippocampus and amygdala as well as total brain, cortical gray matter, white matter, and subcortical gray matter volumes. Relationships between volumes as well as correlations between volumes and cognitive and clinical measures were explored. Ratios of lateral ventricular volume to total brain volume and temporal horn volume to total brain volume were calculated. Volumetric abnormalities were lateralized to the left hemisphere. Volumes of the left temporal horn, and marginally, of the body of the left lateral ventricle were larger, while left amygdala but not hippocampal volume was significantly smaller in CHR participants compared to controls. Total brain volume was also significantly smaller and the ratio of the temporal horn/total brain volume was significantly higher in CHR than in controls. White matter volume correlated positively with higher verbal fluency score while temporal horn volume correlated positively with a greater number of perseverative errors. Together with the finding of larger temporal horns and smaller amygdala volumes in the left hemisphere, these results indicate that the ratio of temporal horns volume to brain volume is abnormal in CHR compared to controls. These abnormalities present in CHR individuals may constitute the biological basis for at least some of the CHR syndrome. [ABSTRACT FROM AUTHOR]

Background: Sym004 is a mixture of two monoclonal antibodies (mAbs), futuximab and modotuximab, targeting non-overlapping epitopes on the epidermal growth factor receptor (EGFR). Previous studies have shown that Sym004 is more efficient at inducing internalization and degradation of EGFR than individual components, which translates into superior cancer cell inhibition. We investigated whether Sym004 induces removal of EGFRvIII and if this removal translates into tumor growth inhibition in hard-to-treat glioblastomas (GBMs) harboring the mutated, constitutively active EGFR variant III (EGFRvIII).Methods: To address this question, we tested the effect of Sym004 versus cetuximab in eight patient-derived GBM xenograft models expressing either wild-type EGFR (EGFRwt) and/or mutant EGFRvIII. All models were tested as both subcutaneous and orthotopic intracranial xenograft models.Results: In vitro studies demonstrated that Sym004 internalized and removed EGFRvIII more efficiently than mAbs, futuximab, modotuximab, and cetuximab. Removal of EGFRvIII by Sym004 translated into significant in vivo anti-tumor activity in all six EGFRvIII xenograft models. Furthermore, the anti-tumor activity of Sym004 in vivo was superior to that of its individual components, futuximab and modotuximab, suggesting a clear synergistic effect of the mAbs in the mixture.Conclusion: These results demonstrate the broad activity of Sym004 in patient-derived EGFRvIII-expressing GBM xenograft models and provide a clear rationale for clinical evaluation of Sym004 in EGFRvIII-positive adult GBM patients. [ABSTRACT FROM AUTHOR]

The water vapor sorption and desorption kinetics were monitored in methyl methacrylate-acrylonitrile-butadiene-styrene copolymer (MABS), bromobutyl, ethylene-propylene-butadiene monomer (EPDM) rubber, and butyl rubber at various temperatures by gravimetric method. Modified thermogravimetric analysis system was built and used. The geometry of the samples was approximating that of the infinite two-sided plane sheet of certain thickness. The coefficients of water vapor diffusion in polymers were deduced by comparing the experimentally monitored kinetics and kinetics computed using diffusion coefficients as adjustable parameters. The activation energies of water vapor diffusion in these polymers were deduced in the temperature range from 30 to 75 °C. The activation energy of diffusion was 45.9 kJ mol in bromobutyl rubber, 46.4 kJ mol in butyl rubber, 60.3 kJ mol in EPDM rubber, and 43.5 kJ mol in MABS. Compensation effect was observed for water vapor diffusion in this series of polymers. Compensation effect parameters were determined to be, a = −23.9/ln(cm s) and b = 0.49/(mol kJ) × ln(cm s), in fair agreement with published data for water diffusion in polymers. [ABSTRACT FROM AUTHOR]

Purpose: Measurement of the second virial coefficient B for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour. In common with all physicochemical chromatographic methods, measuring the dead volume of the SIC packed column is crucial for accurate retention data; this paper examines best practise for dead volume determination. Method: SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements. Results: It was observed that lysozyme and mAb interacted specifically with Toyopearl AF-Formyl dead columns depending upon pH and [NaCl], invalidating their dead volume usage. Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency. Dead volume determinations using dextran MW standards with protein immobilised SIC columns provided dead volume estimates close to those obtained using Toyopearl AF-Amino dead columns. Conclusion: It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns. Two other methods were shown to provide good estimates for the dead volume. [ABSTRACT FROM AUTHOR]

Product yields, efficacy, and safety of monoclonal antibodies (mAbs) are reduced by the formation of higher molecular weight aggregates during upstream processing. In-process characterization of mAb aggregate formation is a challenge since there is a lack of a fast detection method to identify mAb aggregates in cell culture. In this work, we present a rapid method to characterize mAb aggregate-containing Chinese hamster ovary (CHO) cell culture supernatants. The fluorescence dyes thioflavin T (ThT) and 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS) enabled the detection of soluble as well as large mAb aggregates. Partial least square (PLS) regression models were used to evaluate the linearity of the dye-based mAb aggregate detection in buffer down to a mAb aggregate concentration of 2.4 μg mL. Furthermore, mAb aggregates were detected in bioprocess medium using Bis-ANS and ThT. Dye binding to aggregates was stable for 60 min, making the method robust and reliable. Finally, the developed method using 10 μmol L Bis-ANS enabled discrimination between CHO cell culture supernatants containing different levels of mAb aggregates. The method can be adapted for high-throughput screening, e.g., to screen for cell culture conditions influencing mAb product quality, and hence can contribute to the improvement of production processes of biopharmaceuticals in mammalian cell culture. [ABSTRACT FROM AUTHOR]

The bluetongue virus (BTV) NS1 protein is one of the major proteins synthesized during BTV infection and is responsible for the generation of virus-specific tubules. Although some functional and structural studies on the BTV NS1 protein have been reported, there have been no reports describing the linear B-cell epitopes recognized by humoral immune responses published to date. In this study, 25 BTV12 NS1-reactive monoclonal antibodies (MAbs) and polyclonal antisera (polyclonal antibodies, PAbs) were generated and analyzed. We identified 14 linear NS1 epitopes recognized by the PAbs and MAbs using NS1-derived peptides in an enzyme-linked immunosorbent assay. Moreover, we predicted 23 linear B-cell epitopes using the ABCpred online server which employs an artificial neural network. Analysis of the predicted and identified epitopes of NS1 demonstrated the feasibility of B-cell epitope prediction. Sequence alignments indicated that the epitopes recognized by MAbs are highly conserved among BTV serotypes, but not among the other members of the genus Orbivirus, such as the African horse sickness virus (AHSV), epizootic hemorrhagic disease virus (EHDV), and Chuzan disease virus (CV). Importantly, we identified specific MAbs that recognized all BTV serotypes tested as well as MAbs that recognized only BTV12, suggesting that these NS1-specific MAbs could serve as a basis for BTV diagnostic approaches. The generation and identification of NS1 protein epitopes will provide the foundation for further studies about the function and structure of NS1 and novel epitope-based vaccines. [ABSTRACT FROM AUTHOR]

Microneme proteins (MICs) of Eimeria species are critical for motility of the parasite, identification and binding of host cell-surface proteins, invasion of host cells, and intracellular survival. The microneme protein 1 (EtMIC1) and 2 (EtMIC2) from Eimeria tenella have a putative function in parasite adhesion to the host cell to initiate an invasion process. Previous studies indicated that the EtMIC1 and EtMIC2 proteins form a complex that play roles during attachment to and penetration of the host cell. Numerous studies demonstrated that both the EtMIC1 and EtMIC2 are important microneme proteins which are abundantly expressed in sporozoites and schizogony stages. But the expression of EtMIC1 and EtMIC2 in the gametogony stage is unknown. To investigate the precise roles of EtMIC1 and EtMIC2 in host-parasite interactions and expressions in the gametogony stage of E. tenella, we generated five mouse monoclonal antibodies (MAbs) which recognize the EtMIC1 and EtMIC2 proteins and investigated expressions of EtMIC1 and EtMIC2 proteins in later endogenous developmental stages, particularly focused on the gametogony phase using the specific anti-EtMIC1 and anti-EtMIC2 MAbs produced in this work. Our results showed that both EtMIC1 and EtMIC2 proteins are expressed in all developmental stages including the gametogony stage. To our knowledge, this is the first report that the EtMIC1 and EtMIC2 proteins are expressed in the gametogony stage of E. tenella. [ABSTRACT FROM AUTHOR]

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A large-scale serological characterisation of Plum pox virus (PPV) isolates was carried out with 19 monoclonal antibodies (MAbs), including the universal MAb5B and the following strain-specific MAbs: AL (specific to PPV-M), 4DG5 (specific to PPV-D), TUV and AC (specific to PPV-C), and EA24 (specific to PPV-EA). The study involved 108 PPV isolates of different geographical origin (Albania, Bulgaria, Cyprus, Czech Republic, Egypt, France, Germany, Greece, Italy, Hungary, Moldova, Romania, Slovakia, Spain, Turkey and Yugoslavia) and hosts (almond, apricot, peach, plum and cherry). The inter- and intra-strain serological relationships of PPV isolates were evaluated by DASI-ELISA. High serological variability was detected, not only between strains, but also among isolates of the same strain. Computer-assisted analysis of serological data support the hypothesis of the existence of two distinct subclusters, denoted PPV-M1 and PPV-M2, which seem to prevail in Mediterranean and Eastern–Central European countries, respectively. [ABSTRACT FROM AUTHOR]

A reverse trancription-polymerase chain reaction (RT-PCR) assay was devised and shown to be sensitive and reliable for the detection of potato mop-top virus (PMTV) RNA sequences in the flesh of virus infected potato tubers, and in the roots and leaves of soil-bait plants. This assay was compared with an enzyme-linked immunosorbent assay incorporating PMTV specific monoclonal antibodies (TAS-ELISA). The tests were devised to improve the efficiency of detection of viruliferous Spongospora subterranea in agricultural soils, and PMTV in potato tubers. RT-PCR detected PMTV RNA sequences in the roots and leaves of bait plants after three weeks growth in viruliferous soil, three weeks before the bait plants themselves developed symptoms, and two weeks before the virus was detected by TAS-ELISA. Both RT-PCR and TAS-ELISA detected PMTV in the tubers of primary-infected potatoes. RT-PCR and TAS-ELISA were shown to be more sensitive and reliable than conventional baittests and sap inoculation methods for the detection and diagnosis of PMTV. [ABSTRACT FROM AUTHOR]