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In the last decades, novel therapeutics with anabolic bone properties have been developed and are currently used in the management of osteoporosis particularly in patients with high-risk of fragility fractures. These drugs include PTH-Related Analogues, teriparatide and abaloparatide, and the anti-sclerostin agent romosozumab, this latter drug currently approved only in female patients. Their efficacies in preventing fragility fractures are widely demonstrated and their potential serious side effects were progressively downgraded, including risk of malignancies in teriparatide- and cardiovascular events in romosozumab-users, respectively. Further data are warranted about their efficacy in glucocorticoids-induces osteoporosis and fracture healings., Competing Interests: Disclosures The authors have nothing to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Anti-contactin associated protein receptor 2 (CASPR2) encephalitis is a severe autoimmune encephalitis with a variable clinical phenotype including behavioral abnormalities, cognitive decline, epileptic seizures, peripheral nerve hyperexcitability and neuropathic pain. The detailed mechanisms of how CASPR2 autoantibodies lead to synaptic dysfunction and clinical symptoms are largely unknown. Aiming for analyses from the molecular to the clinical level, we isolated antibody-secreting cells from the cerebrospinal fluid of two patients with CASPR2 encephalitis. From these we cloned four anti-CASPR2 human monoclonal autoantibodies (mAbs) with strong binding to brain and peripheral nerves. All were highly hypermutated and mainly of the IgG4 subclass. Mutagenesis studies determined selective binding to the discoidin domain of CASPR2. Surface plasmon resonance revealed affinities with dissociation constants K D in the pico- to nanomolar range. CASPR2 mAbs interrupted the interaction of CASPR2 with its binding partner contactin 2 in vitro and were internalized after binding to CASPR2-expressing cells. Electrophysiological recordings of rat hippocampal slices after stereotactic injection of CASPR2 mAbs showed characteristic afterpotentials following electrical stimulation. In vivo experiments with intracerebroventricular administration of human CASPR2 mAbs into mice and rats showed EEG-recorded brain hyperexcitability but no spontaneous recurrent seizures. Behavioral assessment of infused mice showed a subtle clinical phenotype, mainly affecting sociability. Mouse brain MRI exhibited markedly reduced resting-state functional connectivity without short-term structural changes. Together, the experimental data support the direct pathogenicity of CASPR2 autoantibodies. The minimally invasive EEG and MRI techniques applied here may serve as novel objective, quantifiable tools for improved animal models, in particular for subtle neuropsychiatric phenotypes or repeated measurements., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Previous data suggest a lack of cross-resistance between the gp120-directed attachment inhibitor temsavir (active moiety of fostemsavir) and the CD4-directed post-attachment inhibitor ibalizumab. Recently, analysis of HIV-1 envelopes with reduced sensitivity to both inhibitors was undertaken to determine whether they shared genotypic correlates of resistance. Sequences from 2 envelopes with reduced susceptibility to both agents were mapped onto a temsavir-bound gp120 structure. Residues within 5.0 Å of the temsavir binding site were evaluated using reverse genetics. Broader applicability and contextual determinants of key substitutions were further assessed using envelopes from participants in the phase 3 BRIGHTE study. Temsavir sensitivity was measured by half-maximal inhibitory concentration (IC 50 ) and ibalizumab sensitivity by IC 50 and maximum percent inhibition (MPI). One envelope required substitutions of E113D and T434M for full restoration of temsavir susceptibility. Neither substitution nor their combination affected ibalizumab sensitivity. However, in the second envelope, an E202 substitution (HXB2, T202) was sufficient for observed loss of susceptibility to both inhibitors. One BRIGHTE participant with no ibalizumab exposure had an emergent K202E substitution at protocol-defined virologic failure, with reduced sensitivity to both inhibitors. Introducing T202E into previously susceptible clinical isolates reduced temsavir potency by ≥ 40-fold and ibalizumab MPI from >99% to ∼80%. Interestingly, introduction of the gp120 V5 region from a highly ibalizumab-susceptible envelope mitigated the E202 effect on ibalizumab but not temsavir. A rare HIV-1 gp120 E202 mutation reduced temsavir susceptibility, and depending on sequence context, could result in reduced susceptibility to ibalizumab., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Recently published studies in both murine models and a meta-analysis of non-human primate renal transplant studies showed that anti-CD154 reagents conferred a significant survival advantage over CD40 blockers in both animal models and across multiple organs. Here we sought to compare the induction of donor-reactive forkhead box P3+-induced regulatory T cells (Foxp3 + iTreg) in mice treated with anti-CD154 versus anti-CD40 monoclonal antibodies (mAbs). Results indicated that while treatment with anti-CD154 mAb resulted in a significant increase in the frequency of donor-reactive CD4 + Foxp3 + iTreg following transplantation, treatment with anti-CD40 or Cd40 deficiency failed to recapitulate this result. Because we recently identified CD11b as an alternate receptor for CD154 during alloimmunity, we interrogated the role of CD154:CD11b interactions in the generation of Foxp3 + iTreg and found that blockade of CD11b in Cd40 -/- recipients resulted in increased donor-reactive Foxp3 + iTreg as compared with CD40 deficiency alone. Mechanistically, CD154:CD11b inhibition decreased interleukin (IL)-1β from CD11b + and CD11c + dendritic cells, and blockade of IL-1β synergized with CD40 deficiency to promote Foxp3 + iTreg induction and prolong allograft survival. Taken together, these data provide a mechanistic basis for the observed inferiority of anti-CD40 blockers as compared with anti-CD154 mAb and illuminate an IL-1β-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3 + iTreg during transplantation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mandy L. Ford reports financial support was provided by National Institutes of Health. Mandy L. Ford reports a relationship with Veloxis Pharmaceuticals Inc that includes: consulting or advisory. I am a deputy editor at the American Journal of Transplantation. I am on the Board of Directors of the American Society of Transplantation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society of Transplantation & American Society of Transplant Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Osteosarcoma (OS) is a prevalent primary bone cancer affecting both humans and canines. This study describes initial insights into the interaction of the human monoclonal antibody IF3 to an insulin-like growth factor 2 receptor (IGF2R) radiolabeled with either alpha-emitting Actinium-225 ( 225 Ac) or beta-emitting Lutetium-177 ( 177 Lu) radionuclides with the OS cells and tumor microenvironment (TME) in experimental human and canine OS., Basic Procedures: SCID mice bearing canine Gracie or human OS-33 OS tumors were treated with 177 Lu- or 225 Ac-labeled IF3 antibody, sacrificed at 24, 72 or 168 h post-treatment and their tumors were analyzed by immunohistochemistry (IHC) for the presence of OS cells, various elements of TME as well as for the double DNA strand breaks with γH2AX and caspase 3 assays., Main Findings: IHC revealed a reduction in IGF2R-positive OS cells and OS stem cell populations post therapy with 225 Ac- and 177 Lu-labeled IF3 antibody. Notably, radiolabeled IF3 antibody effectively diminished pro-tumorigenic M2 macrophages, highlighting its therapeutic promise. The study also unveiled varied responses of natural killer (NK) cells and M1 macrophages, shedding light on the intricate TME interplay. Time-dependent increase in γ-H2AX staining in canine Gracie and human OS-33 tumors treated with [ 177 Lu]Lu-IF3 and [ 225 Ac]Ac-IF3 was observed at 24 and 72 h post-RIT., Principal Conclusions: These findings suggest that radiolabeled antibodies offer a hopeful avenue for personalized OS treatment, emphasizing the importance of understanding their impact on the TME and potential synergies with immunotherapy., Competing Interests: Declaration of competing interest I, Ekaterina Dadachova, serve as an associate editor of NMB. I declare that I do not have any conflict of interest by being a senior author on the submitted manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Invasive Aspergillosis is a high-risk illness with a high death rate in immunocompromised people due to a lack of early detection and timely treatment. Based on immunology study, we achieved an efficient production of anti-galactomannan antibody by Chinese hamster ovary (CHO) cells and applied it to time-resolved fluoroimmunoassay for Aspergillus galactomannan detection. We first introduced dual promoter expression vector into CHO host cells, and then applied a two-step screening strategy to screen the stable cell line by methionine sulfoximine pressurization. After amplification and fermentation, antibody yield reached 4500 mg/L. Then we conjugated the antibodies with fluorescent microspheres to establish a double antibody sandwich time-resolved fluoroimmunoassay, which was compared with the commercial Platelia™ Aspergillus Ag by clinical serum samples. The preformed assay could obtain the results in less than 25 min, with a limit of detection for galactomannan of approximately 1 ng/mL. Clinical results of the two methods showed that the overall percent agreement was 97.7% (95% CI: 96.6%-98.4%) and Cohen's kappa coefficient was 0.94. Overall, the assay is highly consistent with commercial detection, providing a more sensitive and effective method for the rapid diagnosis of invasive aspergillosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The surge in opioid-related deaths, driven predominantly by fentanyl and its synthetic derivatives, has become a critical public health concern, which is particularly evident in the United States. While the situation is less severe in Europe, the European Monitoring Centre for Drugs and Drug Addiction reports a rise in drug overdose deaths, with emerging concerns about the impact of fentanyl-related molecules. Synthetic opioids, initially designed for medical use, have infiltrated illicit markets due to their low production costs and high potency, with carfentanil posing additional threats, including potential chemical weaponization. Existing overdose mitigation heavily relies on naloxone, requiring timely intervention and caregiver presence, while therapeutic prevention strategies face many access challenges. To provide an additional treatment option, we propose the use of a fentanyl-specific monoclonal antibody (mAb), as a non-opioid method of prophylaxis against fentanyl and carfentanil. This mAb shows protection from opioid effects in a pre-clinical murine model. mAbs could emerge as a versatile countermeasure in civilian and biodefense settings, offering a novel approach to combat opioid-associated mortality., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Joseph Verdi reports financial support was provided by National Institute on Drug Abuse. Nina Papavasiliou reports financial support was provided by Helmholtz Foundation. Erec Stebbins reports financial support was provided by Helmholtz Foundation. Joseph Verdi reports a relationship with Hepione Therapeutics Inc. that includes: employment and equity or stocks. Nina Papavasiliou reports a relationship with Hepione Therapeutics Inc. that includes: equity or stocks. Erec Stebbins reports a relationship with Hepione Therapeutics Inc. that includes: board membership and equity or stocks. Yosip Kelemen reports a relationship with Hepione Therapeutics Inc. that includes: employment. Joseph Verdi reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Nina Papavasiliou reports a relationship with Panosome GmbH that includes: board membership and equity or stocks. Erec Stebbins reports a relationship with Panosome GmbH that includes: board membership and equity or stocks. Anastasia Gkeka reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Katharina Urban reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Monica Chandra reports a relationship with Panosome GmbH that includes: employment and equity or stocks. Dennis Greiner reports a relationship with Panosome GmbH that includes: employment. Sandra Ruf reports a relationship with Panosome GmbH that includes: equity or stocks. Selina Pollich reports a relationship with Panosome GmbH that includes: employment. Joseph Verdi has patent pending to Hepione Therapeutics Inc. Nina Papavasiliou has patent pending to Hepione Therapeutics Inc. Erec Stebbins has patent pending to Hepione Therapeutics Inc. Yosip Kelemen has patent pending to Hepione Therapeutics Inc. Marco Pravetoni has patent pending to University of Minnesota. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The emergence of COVID-19 caused by the coronavirus SARS-CoV-2 has prompted a global pandemic that requires continuous research and monitoring. This study presents a design of an electrochemical biosensing platform suitable for the evaluation of monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid (N) protein. Screen-printed carbon electrodes (SPCE) modified with gold nanostructures (AuNS) were applied to design a versatile and sensitive sensing platform. Electrochemical techniques, including electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), were used to investigate the interactions between immobilised recombinant N (rN) protein and several monoclonal antibodies (mAbs). The electrochemical characterisation of SPCE/AuNS/rN demonstrated a successful immobilisation of rN, enhancing the electron transfer kinetics. Affinity interactions between immobilised rN and four mAbs (mAb-4B3, mAb-4G6, mAb-12B2, and mAb-1G5) were explored. Although mAb-4B3 showed some non-linearity, the other monoclonal antibodies exhibited specific and well-defined interactions followed by the formation of an immune complex. The biosensing platform demonstrated high sensitivity in the linear range (LR) from 0.2 nM to 1 nM with limits of detection (LOD) ranging from 0.012 nM to 0.016 nM for mAb-4G6, mAb-12B2, and mAb-1G5 and limits of quantification (LOQ) values ranging from 0.035 nM to 0.139 nM, as determined by both EIS and SWV methods. These results highlight the system's potential for precise and selective detection of monoclonal antibodies specific to the rN. This electrochemical biosensing platform provides a promising route for the sensitive and accurate detection of monoclonal antibodies specific to the rN protein., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Monkeypox (mpox) is spreading around the world, and its rapid diagnosis is of great significance. In the present study, a rapid and sensitive fluorescent chromatography assisted with cloud system was developed for point-of-care diagnosis of mpox. To screen high affinity antibodies, nanoparticle antigen AaLS-A29 was generated by conjugating A29 onto scaffold AaLS. Immunization with AaLS-A29 induced significantly higher antibody titers and monoclonal antibodies were generated with the immunized mice. A pair of monoclonal antibodies, MXV 14 and MXV 15, were selected for fluorescence chromatography development. The Time-Resolved Fluorescence Immunoassay (TRFIA) was used to develop the chromatography assay. After optimization of the label and concentration of antibodies, a sensitive TRFIA assay with detection limit of 20 pg/mL and good repeatability was developed. The detection of the surrogate Vaccinia virus (VACA) strain Tian Tan showed that the TRFIA assay was more sensitive than the SYBR green I based quantitative PCR. In real samples, the detection result of this assay were highly consistent with the judgement of Quantitative Real-Time PCR (Concordance Rate = 90.48%) as well as the clinical diagnosis (Kappa Value = 0.844, P < 0.001). By combining the portable detection and online cloud system, the detection results could be uploaded and shared, making this detection system an ideal system for point-of-care diagnosis of mpox both in field laboratory and outbreak investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

High-performance antibodies are core reagents for highly sensitive immunoassays. Herein, based on a novel hapten, a hybridoma secreting the high-affinity anti-ethirimol monoclonal antibody (mAb-14G5F6) was isolated with an IC 50 value of 1.35 μg/L and cross-reactivity below 0.20% for 13 analogs. To further address the challenge of hybridoma preservation and antibody immortalization, a recombinant full-length antibody (rAb-14G5F6) was expressed using the HEK293(F) expression system based on the mAb-14G5F6 gene. The affinity, specificity, and tolerance of rAb-14G5F6, as characterized by indirect competitive enzyme-linked immunosorbent assay and noncompetitive surface plasmon resonance, exhibited high concordance with those of mAb-14G5F6. Further immunoassays based on rAb-14G5F6 were developed for irrigation water and strawberry fruit with limits of detection of 0.0066 and 0.036 mg/kg, respectively, recoveries of 80100%, and coefficients of variation below 10%. Furthermore, homology simulation and molecular docking revealed that GLU(L40), GLY(L107), GLY(H108), and ASP(H114) play important roles in forming hydrogen bonds and pi-anion ionic bonds between rAb-14G5F6 and ethirimol, resulting in the high specificity and affinity of rAb-14G5F6 for ethirimol, with a K D of 5.71 × 10 -10 mol/L. Overall, a rAb specific for ethirimol was expressed successfully in this study, laying the groundwork for rAb-based immunoassays for monitoring fungicide residues in agricultural products and the environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Contamination of oilfield chemicals (OFCs) by benzo[a]pyrene (B[a]P) is increasingly becoming a severe environmental security issue. There is an urgent need to develop a rapid and accurate method for B[a]P detection in OFCs. In this study, B[a]P hapten was designed using computer aided molecular design. A high-affinity, specific, and matrix-insensitive monoclonal antibody (mAb) with IC 50 values of 6.77 ng/mL was obtained. Based on this mAb, we developed a rapid gold nanoparticle-based immunochromatographic strip assay (GICA) with double T-line mode for on-site detection of B[a]P in OFCs samples. The GICA exhibited excellent detection performance in OFCs samples with strong acidity, strong alkalinity, and deep color. Under optimal conditions, the proposed method detected B[a]P in OFCs at 0.42-300 mg/kg, and limit of detection was 0.23-1.07 mg/kg. The recovery rate was 88-106% with a coefficient of variation of 1.46-6.35%. Confirmed by natural positive OFCs samples and high-performance liquid chromatography, this GICA is accurate and reliable, with great potential for rapid and cost-effective on-site detection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Development of assays to reliably identify and characterize anti-drug antibodies (ADAs) depends on positive control anti-idiotype (anti-id) reagents, which are used to demonstrate that the standards recommended by regulatory authorities are met. This work employs a set of therapeutic antibodies under clinical development and their corresponding anti-ids to investigate how different positive control reagent properties impact ADA assay development. Positive controls exhibited different response profiles and apparent assay analytical sensitivity values depending on assay format. Neither anti-id affinity for drug, nor sensitivity in direct immunoassays related to sensitivity in ADA assays. Anti-ids were differentially able to detect damage to drug conjugates used in bridging assays and were differentially drug tolerant. These parameters also failed to relate to assay sensitivity, further complicating selection of anti-ids for use in ADA assay development based on functional characteristics. Given this variability among anti-ids, alternative controls that could be employed across multiple antibody drugs were investigated as a more uniform means to define ADA detection sensitivity across drug products and assay protocols, which could help better relate assay results to clinical risks of ADA responses. Overall, this study highlights the importance of positive control selection to reliable detection and clinical interpretation of the presence and magnitude of ADA responses., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Drug conjugation to an antibody can affect its stability, which depends on factors such as the conjugation technique used, drug-linker properties, and stress encountered. This study focused on the effects of agitation stress on the physical stability of two lysine (ADC-K) and two interchain cysteine (ADC-C) conjugates of an IgG 1 monoclonal antibody (mAb) linked to either ∼4 MMAE or DM1 payloads. During agitation, all antibody-drug conjugates (ADCs) exhibited higher aggregation than the mAb, which was dependent on the conjugation technique (aggregation of ADC-Ks > ADC-Cs) and drug-linker (aggregation of ADCs with MMAE > ADCs with DM1). The aggregation propensities correlated well with higher self-interaction, hydrophobicity, and surface activity of ADCs relative to the mAb. The intermediate reduced mAb (mAb-SH) showed even higher aggregation than the final product ADC-Cs. However, blocking mAb-SH's free thiols with N-ethylmaleimide (NEM) strongly reduced its aggregation, suggesting that free thiols should be minimized in cysteine ADCs. Further, this study demonstrates that a low-volume surface tension method can be used for estimating agitation-induced aggregation of ADCs in early development phases. Identifying liabilities to agitation stress and their relationship to biophysical properties may help optimize ADC stability., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems., Competing Interests: Declaration of competing interest All authors have read and approve of this manuscript and further affirm that the information presented is true and correct and are willing to take public responsibility for the manuscript. The authors declare that they have no conflict of interest with the contents of the article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Monoclonal antibodies (mAbs) continue to dominate the biopharmaceutical industry. Certain mAbs are prone to fragmentation and clipping and in these cases, adequate removal of these species is critical during manufacturing. Fragments can be generated during fermentation, purification, storage, formulation, and administration. Their addition to the acidic charge-variant of the purified mAb has been reported to decrease stability and potency of the final product. However, contrary to mAb aggregation, manufacturers have not given much attention to removal of fragments and clipped species and as a result most conventional mAb platforms offer at best limited capabilities for their removal. In this study, we propose a novel purification platform that uses multimodal chromatography and achieves complete removal of a range of mAb fragments and clipped products (25-120 kDa). The utility of the platform has been successfully demonstrated for 2 IgG1s and 2 IgG4s. Further, adequate removal of the various host cell impurities such as host cell proteins (<10 ppm) and host cell DNA (<5 ppb) has been achieved. Finally, the platform was able to deliver adequate removal of high molecular weight impurities (<1 %) and a 30 % clearance of the acidic charge variant. The proposed single step has been shown to deliver what the polishing chromatography and intermediate purification chromatography steps deliver in a traditional mAb platform., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Anurag Singh Rathore, PhD reports financial support was provided by India Ministry of Science & Technology Department of Biotechnology. Vineela Peruri reports financial support was provided by Council of Scientific & Industrial Research., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gardenia jasminoides Ellis, a plant widely used in traditional medicine, is known for its array of biological activities. A key bioactive compound, geniposide (GE), an iridoid glycoside, significantly contributes to the medicinal properties of the plant, with potential side effects. Thus, a reliable and efficient method for GE detection is required to ensure the quality of medicinal-grade G. jasminoides Ellis. This study developed such a method by first synthesizing GE-bovine serum albumin conjugates to function as immunizing agents in mice. This led to the production of a monoclonal antibody (mAb 3A6) against GE from the fusion of splenocytes from immunized mice with myeloma cells (P3U1), resulting in a hybridoma that produces mAb 3A6. Thereafter, we developed a mAb 3A6-based indirect competitive enzyme-linked immunosorbent assay (icELISA). The icELISA exhibited satisfactory sensitivity (0.391-12.5 μg/ml) and repeatability (coefficients of variation <10%). The accuracy of this method was validated through a spike-recovery assay (recovery of 101-112%). Furthermore, the icELISA was employed to determine the GE content in plant and Kampo medicine samples. The GE content positively correlated with those determined by high-performance liquid chromatography-ultraviolet. The proposed icELISA is rapid, cost-effective, and reliable for high-throughput GE detection in G. jasminoides Ellis, thereby contributing to the improved quality control and standardization of this valuable medicinal plant., Competing Interests: Declaration of competing interest The authors declare no competing interests or conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The PD-1/PD-L1 protein-protein interaction (PPI) controls an adaptive immune resistance mechanism exerted by tumor cells to evade immune responses. The large-molecule nature of current commercial monoclonal antibodies against this PPI hampers their effectiveness by limiting tumor penetration and inducing severe immune-related side effects. Synthetic small-molecule inhibitors may overcome such limitations and have demonstrated promising clinical translation, but their design is challenging. Microbial natural products (NPs) are a source of small molecules with vast chemical diversity that have proved anti-tumoral activities, but which immunotherapeutic properties as PD-1/PD-L1 inhibitors had remained uncharacterized so far. Here, we have developed the first cell-based PD-1/PD-L1 blockade reporter assay to screen NPs libraries. In this study, 6000 microbial extracts of maximum biosynthetic diversity were screened. A secondary metabolite called alpha-cyclopiazonic acid (α-CPA) of a bioactive fungal extract was confirmed as a new PD-1/PD-L1 inhibitor with low micromolar range in the cellular assay and in an additional cell-free competitive assay. Thermal denaturation experiments with PD-1 confirmed that the mechanism of inhibition is based on its stabilization upon binding to α-CPA. The identification of α-CPA as a novel PD-1 stabilizer proves the unprecedented resolution of this methodology at capturing specific PD-1/PD-L1 PPI inhibitors from chemically diverse NP libraries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

African Swine Fever Virus (ASFV) is a highly contagious pathogen posing a serious threat to the global swine industry. Despite this, there is currently no effective vaccine against this virus. Within ASFV's core shell structure, p37, a product of polyprotein pp220, shares sequence similarity with SUMO-1 proteases. Localization studies show p37 in various nuclear regions during early infection, shifting to the cytoplasm later on. Research indicates active export of p37 from the nucleus, mediated by CRM1-dependent and -independent pathways. Hydrophobic amino acids in p37 are crucial for these pathways, highlighting their importance throughout the ASFV replication cycle. Additionally, p37 serves as the first nucleocytoplasmic shuttle protein encoded by ASFV, participating in the intranuclear material transport process during ASFV infection of host cells. In this study, we successfully screened five murine monoclonal antibodies targeting p37. Through the truncated expression method, we identified four dominant antigenic epitopes of p37 for the first time. Furthermore, utilizing alanine scanning technology, we determined the key amino acid residues for each epitope. This research not only provides essential information for a deeper understanding of the protein's function but also establishes a significant theoretical foundation for the design and development of ASFV vaccines., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

In recent years, tumor immunotherapy, aimed at increasing the activity of immune cells and reducing immunosuppressive effects, has attracted wide attention. Among them, immune checkpoint blocking (ICB) is the most commonly explored therapeutic approach. All approved immune checkpoint inhibitors (ICIs) are clinically effective monoclonal antibodies (mAbs). Compared with biological agents, small-molecule drugs have many unique advantages in tumor immunotherapy. Therefore, they also play an important role. Immunosuppressive signals such as PD-L1, IDO1, and TGF-β, etc. overexpressed in tumor cells form the tumor immunosuppressive microenvironment. In addition, the efficacy of multi-pathway combined immunotherapy has also been reported and verified. Here, we mainly reviewed the mechanism of tumor immunotherapy, analyzed the research status of small-molecule modulators, and discussed drug candidates' structure-activity relationship (SAR). It provides more opportunities for further research to design more immune small-molecule modulators with novel structures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that causes huge economic losses to the global pig industry. Nonstructural protein 7α (NSP7α) of PRRSV is highly conserved among different lineages of PRRSV and could be a potential target for the development of detection methods. In this study, NSP7α was expressed in prokaryote (Escherichia coli) and purified. An NSP7α-ab-ELISA detection method was established, the NSP7α-ab-ELISA has 93.1 % coincidence rate with IDEXX PRRS X3 ab test kit. NSP7α antibody was detected in pig serum by ELISA 14 days following PRRSV infection. Three monoclonal antibodies (4H9, 3F2, and C10) against NSP7α prepared by a hybridoma technique were used for epitope mapping by indirect immunofluorescence. The 4H9, 3F2, and C10 antibodies all recognized the C-terminal 72-149 amino acid region of NSP7α. 4H9 reacted with amino acids 135-143, but 3F2 and C10 did not react with any truncated polypeptide. In addition, by using the monoclonal antibodies, NSP7α was localized solely in the cytoplasm, while the N protein was distributed in the cytoplasm and nucleus. The collective findings of the antigenicity and epitope of NSP7α will be helpful for understanding the antigenicity of NSP7α and developing PRRSV diagnostic methods., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tongqing An reports financial support was provided by the Heilongjiang Provincial Natural Science Foundation of China. Tongqing An reports financial support was provided by Heilongjiang Key Research and Development Program. Tongqing An reports financial support was provided by the National Natural Science Foundation of China., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: This study aimed to investigate whether genetic alterations in PI3KCA and the cell cycle pathways influence the efficacy of durvalumab, an immune checkpoint inhibitor, in patients with head and neck squamous cell carcinoma (HNSCC) who had previously failed platinum-based treatment., Materials and Methods: We obtained data from a phase II umbrella trial of patients with HNSCC who failed platinum-based treatment (TRIUMPH, NCT03292250). Patients receiving durvalumab treatment comprised those with PIK3CA alterations (Group A), those with cell cycle pathway alterations such as CDKN2A (Group B), and those with no druggable genetic alterations (Group C). We analyzed the overall response rate (ORR), progression-free survival (PFS), and overall survival (OS) in each group and evaluated the potential predictive factors for durvalumab., Results: We analyzed the data of 87 patients: 18, 12, and 57 in groups A, B, and C, respectively. The ORRs were 27.8 %, 8.3 %, and 15.8 % in Groups A, B, and C, respectively (P = 0.329), and the median PFS for each group was 2.3, 1.6, and 1.7 months, respectively, with no significant differences between the groups (P = 0.24). Notably, patients with lower neutrophil-lymphocyte ratio (NLR) (≤5.8) had longer PFS (median, 2.8 vs 1.6 months, P < 0.001), while those with lower platelet-lymphocyte ratio (PLR) (≤491.2) exhibited longer PFS (median, 1.8 vs 1.2 months, P < 0.001)., Conclusion: Durvalumab's efficacy was similar, irrespective of the presence of PIK3CA or cell cycle pathway genetic alterations in patients with platinum-resistant HNSCC. The NLR and PLR may be promising predictive biomarkers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors certified that they have no conflict of interest (COI) directly related to this report. Broad information about their COI is presented. BK received research funding from MSD, AstraZeneca and Ono Pharmaceutical Co., Ltd., and has served as an advisor for Handok, NeoImmuneTec, Trialinformatics and ImmuneOncia outside of the current work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Monoclonal antibodies have revolutionized the treatment of human diseases, which has made them the fastest-growing class of therapeutics, with global sales expected to reach $346.6 billion USD by 2028. Advances in antibody engineering and development have led to the creation of increasingly sophisticated antibody-based therapeutics (e.g. bispecific antibodies and chimeric antigen receptor T cells). However, approaches for antibody discovery have remained comparatively grounded in conventional yet reliable in vitro assays. Breakthrough developments in high-throughput single B-cell sequencing and immunoglobulin proteomic serology, however, have enabled the identification of high-affinity antibodies directly from endogenous B cells or circulating immunoglobulin produced in vivo. Moreover, advances in artificial intelligence offer vast potential for antibody discovery and design with large-scale repertoire datasets positioned as the optimal source of training data for such applications. We highlight advances and recent trends in how these technologies are being applied to antibody repertoire analysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension ( 1 D) and WCX in the second dimension ( 2 D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: R. Kenneth Marcus reports financial support was provided by National Science Foundation. Sarah W. Harcum reports financial support was provided by National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although antibody fragments are a critical impurity to remove from process streams, few platformable purification techniques have been developed to this end. In this work, a novel size-exclusion-mixed-mode (SEMM) resin was characterized with respect to its efficacy in mAb fragment removal. Inverse size-exclusion chromatography showed that the silica-based resin had a narrow pore size distribution and a median pore radius of roughly 6.2 nm. Model-based characterization was carried out with Chromatography Analysis and Design Toolkit (CADET), using the general rate model and the multicomponent Langmuir isotherm. Model parameters were obtained from fitting breakthrough curves, performed at multiple residence times, for a mixture of mAb, aggregates, and an array of fragments (varying in size). Accurate fits were obtained to the frontal chromatographic data across a range of residence times. Model validation was then performed with a scaled-up column, altering residence time and feed composition from the calibration run. Accurate predictions were obtained, thereby illustrating the model's interpolative and extrapolative capabilities. Additionally, the SEMM resin achieved 90% mAb yield, 37% aggregate removal, 29% [Formula: see text] removal, 54% Fab/Fc removal, 100% Fc fragments removal, and a productivity of 72.3 g mAbL×h. Model predictions for these statistics were all within 5%. Simulated batch uptake experiments showed that resin penetration depth was directly related to protein size, with the exception of the aggregate species, and that separation was governed by differential pore diffusion rates. Additional simulations were performed to characterize the dependence of fragment removal on column dimension, load density, and feed composition. Fragment removal was found to be highly dependent on column load density, where optimal purification was achieved below 100 mg protein/mL column. Furthermore, fragment removal was dependent on column volume (constant load mass), but agnostic to whether column length or diameter was changed. Lastly, the dependence on feed composition was shown to be complex. While fragment removal was inversely related to fragment mass fraction in the feed, the extent depended on fragment size. Overall, the results from this study illustrated the efficacy of the SEMM resin in fragment and aggregate removal and elucidated relationships with key operational parameters through model-based characterization., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Steven M. Cramer reports financial support was provided by National Institute for Innovation in Manufacturing Biopharmaceuticals. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Forced degradation, also known as stress testing, is used throughout pharmaceutical development for many purposes including assessing the comparability of biopharmaceutical products according to ICH Guideline Q5E. These formal comparability studies, the results of which are submitted to health authorities, investigate potential impacts of manufacturing process changes on the quality, safety, and efficacy of the drug. Despite the wide use of forced degradation in comparability assessments, detailed guidance on the design and interpretation of such studies is scarce. The BioPhorum Development Group is an industry-wide consortium enabling networking and sharing of common practices for the development of biopharmaceuticals. The BioPhorum Development Group Forced Degradation Workstream recently conducted several group discussions and a benchmarking survey to understand current industry approaches for the use of forced degradation studies to assess comparability of protein-based biopharmaceuticals. The results provide insight into the design of forced degradation studies, analytical characterization and testing strategies, data evaluation criteria, as well as some considerations and differences for non-platform modalities (e.g., non-traditional mAbs). This article presents survey responses from several global companies of various sizes and provides an industry perspective and experience regarding the practicalities of using forced degradation to assess comparability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by a concerning rise in prevalence. It is projected that the number of affected individuals will reach a staggering 150 million by 2050. While recent advancements in monoclonal antibodies targeting Aβ have shown some clinical effects, there is an urgent need for improved therapies to effectively address the impeding surge of AD patients worldwide. To achieve this, a deeper understanding of the intricate mechanisms underlying the disease is crucial. In recent years, mounting evidence has underscored the vital role of the innate immune system in AD pathology. However, limited findings persist regarding the involvement of the adaptive immune system. Here, we report on the impact of the adaptive immune system on various aspects of AD by using App NL-G-F mice crossed into a Rag2 -/- background lacking mature adaptive immune cells. In addition, to simulate the continuous exposure to various challenges such as infections that is commonly observed in humans, the innate immune system was activated through the repetitive induction of peripheral inflammation. We observed a remarkably improved performance on complex cognitive tasks when a mature adaptive immune system is absent. Notably, this observation is pathologically associated with lower Aβ plaque accumulation, reduced glial activation, and better-preserved neuronal networks in the mice lacking a mature adaptive immune system. Collectively, these findings highlight the detrimental role of the adaptive immune system in AD and underscore the need for effective strategies to modulate it for therapeutic purposes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Competing Interests: Declaration of Competing Interest CG has received grants from Italian Ministry of Health and BMS and Honoraria for presentations or lectures from AstraZeneca, BMS, Lilly, MSD, Roche, Sanofi, Takeda, Thermo Fisher; ML has received personal fees (advisory role and/or speaker honoraria) from Roche, Takeda, Sandoz, Eli Lilly, Pfizer, AstraZeneca, Novartis Exact Sciences and Ipsen; LDM has reported consulting fees from Roche, Novartis, MSD, Pfizer, Ipsen, AstraZeneca, Genomic Health, Eli Lilly, Seattle Genetics, Eisai, Pierre Fabre, and Daiichi Sankyo, speaker Honoraria from Roche, Novartis, Eli Lilly and MSD, travel grants from Roche, Pfizer and Celgene; FS has reported Honoraria for presentations or lectures from Sanofi Genzyme, Roche, BMS, Novartis, Merk, Sun Pharma, MSD, Pierre Fabre and for advisory board for Novartis, Philogen, SunPharma, MSD.

Since 2011, PRV has resurged in China and is characterized by a mutated strain with significant alterations in antigenicity and virulence. Therefore, we hypothesized that antibody detection kits based on classic PRV strains may have limitations in detecting PRV variants. For more sensitive antibody detection of PRV variants, two MABs targeting the gB and gE proteins were developed. IFA revealed that these MABs exhibited strong reactivity toward both classic and variant PRV strains. MAB-gE recognizes a novel conserved linear B-cell epitope ( 41 PSAEVWD 47 ), while MAB-gB recognizes a conformational B-cell epitope. The binding of both MABs was effectively inhibited in the PRV-positive pig blood samples. Accordingly, we established blocking-ELISAs to detect anti-PRV gB and gE antibodies, which achieved higher sensitivity than commercial kits. Moreover, the clinical serum samples results of our method and that of IFA were in high agreement, and our test results had a higher coincidence rate than that of a commercial kit. Assessing antibody levels by our methods at various times following immunization and challenge accurately reflected the trend of antibody-level changes and revealed the conversion to positive antibody status before the commercial kit. Our method is crucial for monitoring PRV infections, assessing immune responses, and controlling disease., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Significant challenges to poultry health are posed by chicken anemia virus (CAV), which induces immunosuppression and causes increased susceptibility to secondary infections. The effective management and containment of CAV within poultry stocks require precise and prompt diagnosis. However, a deficiency persists in the availability of low-cost, rapid, and portable CAV detection devices. In this study, an immunochromatographic lateral-flow test strip-based assay was developed for CAV detection using in-house generated monoclonal antibodies (MABs) against CAV viral protein 1 (VP1). The recombinant truncated VP1 protein (Δ60VP1), with amino acid residues 1 to 60 of the native protein deleted, was produced via a prokaryotic expression system and utilized for immunizing BALB/c mice. Subsequently, high-affinity MABs against Δ60VP1 were generated and screened using conventional hybridoma technology combined with serial dilution assays. Two MABs, MAB1, and MAB3, both binding to distinct epitopes of Δ60VP1, were selected for the development of a lateral-flow assay. Sensitivity analysis demonstrated that the Δ60VP1 antigen could be detected by our homemade lateral-flow assay at concentrations as low as 625 ng/mL, and this sensitivity was maintained for at least 6 mo. The assay exhibited high specificity, as evidenced by its lack of reactivity with surrogate recombinant proteins and the absence of cross-reactivity with other chicken viruses and viral antigens. Comparative analysis with quantitative PCR data demonstrated substantial agreement, with a Kappa coefficient of 0.66, utilizing a sample set comprising 305 clinical chicken serum samples. In conclusion, the first lateral-flow assay for CAV detection was developed in this study, utilizing 2 specific anti-VP1 MABs. It is characterized by simplicity, rapidity, sensitivity, and specificity., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Consolidation durvalumab following platinum-based chemoradiotherapy (CRT) significantly improved overall survival for patients with unresectable stage III non-small cell lung cancer (NSCLC) in the PACIFIC trial. However, older patients were underrepresented in PACIFIC, and subsequent analyses suggested trends toward poorer survival and increased toxicity in patients aged ≥70 years old. We assessed the effectiveness and safety of consolidation durvalumab following CRT in older Australian patients with unresectable stage III NSCLC., Materials and Methods: This retrospective observational study was conducted across seven sites in Sydney, Australia between January 2018 and September 2021. All adult patients with unresectable stage III NSCLC who received platinum-based chemoradiotherapy followed by at least one cycle of consolidation durvalumab were included. Older patients were defined as being ≥70 years old., Results: Of 152 patients included in the analysis, 42.8% (n = 67) patients were 70 years or older. Median follow-up was 26.1 months. The two-year overall survival and median PFS was similar between older and younger patients. At two years, 74.8% (95% confidence interval [CI]: 65.4-84.2%) of patients <70 years old and 65.2% (95% CI: 53.4-77.0%) of older patients were alive (p = 0.07; hazard ratio [HR] 1.64, 95% CI: 0.95-2.81). Median progression-free survival (PFS) in patients <70 years was 30.3 months (95% CI: 22.2-38.4 months) compared with 26.7 months (95% CI: 12.8-40.6 months) in older patients (p = 0.22; HR 1.46, 95% CI: 0.80-2.65). Toxicity was also similar, with 11.5% of patients <70 years old and 18.5% of older patients experiencing grade 3-4 adverse events (AEs; p = 0.23); 16.1% and 24.6% of the patients, respectively, discontinued treatment due to toxicity (p = 0.19). Grade 3-4 AEs and treatment discontinuation were associated with Charlson Comorbidity Index >5 (p = 0.011) and chronic obstructive pulmonary disease diagnosis at presentation (p = 0.002), respectively., Discussion: Older Australian patients receiving consolidation durvalumab following CRT experienced comparable outcomes to their younger peers. Comorbidity burden may be more important determinants of treatment tolerance than chronological age., Competing Interests: Declaration of Competing Interest, (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: AB reports consulting fees from Kabi, Roche Chugai, GlaxoSmithKline, AbbVie, Novartis, and Boehringer Ingelheim; research grants from Boehringer Ingelheim, ANR, and Horizon Europe outside the scope of this Comment; and is an unpaid co-chair of the French National Network on Rare Autoimmune and Autoinflammatory Diseases. All other authors declare no competing interests.

Background: CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (CHAPLE) is an ultra-rare genetic disorder characterised by intestinal lymphatic damage, lymphangiectasia, and protein-losing enteropathy caused by overactivation of the complement system. We assessed the efficacy and safety of pozelimab, an antibody blocking complement component 5., Methods: This open-label, single-arm, historically controlled, multicentre phase 2 and 3 study evaluated ten patients with CHAPLE disease. This study was conducted at three hospitals in Thailand, Türkiye, and the USA. Patients aged 1 year or older with a clinical diagnosis of CHAPLE disease and a CD55 loss-of-function variant identified by genetic analysis and confirmed by flow cytometry or western blot of CD55 from peripheral blood cells were eligible for this study. Patients received a single intravenous loading dose of pozelimab 30 mg per kg of bodyweight, followed by a once-per-week subcutaneous dose over the treatment period based on bodyweight at a concentration of 200 mg/mL as either a single injection (<40 kg bodyweight) or two injections (≥40 kg bodyweight). The primary endpoint was proportion of patients with serum albumin normalisation with an improvement in active clinical outcomes and no worsening in inactive clinical outcomes (frequency of problematic abdominal pain, bowel movement frequency, facial oedema severity, and peripheral oedema severity) at week 24 compared with baseline, assessed in the full analysis set. This study is registered with ClinicalTrials.gov (NCT04209634) and is active but not recruiting., Findings: 11 patients were recruited between Jan 27, 2020, and May 12, 2021, ten of which were enrolled in the study and included in the analysis populations. The efficacy data corresponded to all patients completing the week 48 assessment and having at least 52 weeks of treatment exposure, and the safety data included an additional 90 days of follow-up and corresponded to all patients having at least 72 weeks of treatment. Patients were predominantly paediatric (with a median age of 8·5 years), and originated from Türkiye, Syria, Thailand, and Bolivia. Patients had markedly low weight-for-age and stature-for-age at baseline, and mean albumin at baseline was 2·2 g/dL, which was considerably less than the local laboratory reference range. After pozelimab treatment, all ten patients had serum albumin normalisation and improvement with no worsening in clinical outcomes. There was a complete inhibition of the total complement activity. Nine patients had adverse events; two were severe events, and one patient had an adverse event considered related to pozelimab., Interpretation: Pozelimab inhibits complement overactivation and resolves the clinical and laboratory manifestations of CHAPLE disease. Pozelimab is the only currently approved therapeutic drug for patients with this life-threatening, ultra-rare condition. In patients with protein-losing enteropathy where known causes have been excluded, testing for a CD55 deficiency should be contemplated. A diagnosis of CHAPLE disease should lead to early consideration of treatment with pozelimab., Funding: Regeneron Pharmaceuticals and the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health., Competing Interests: Declaration of interests AO is a consultant and steering committee member for Regeneron Pharmaceuticals; received sample analysis support for a previous collaborative study (https://doi.org/10.1038/s41590-020-00830-z) from Regeneron Pharmaceuticals; and has a pending patent on component 5 inhibitor treatment in CHAPLE disease. VC, NS, and KS received support to conduct the study and received provision of the investigational product from Regeneron Pharmaceuticals. JJJ, TB, and LP are Regeneron Pharmaceuticals employees and stockholders. KAM is a Regeneron Pharmaceuticals employee and stockholder and has both pending and issued patents with Regeneron Pharmaceuticals. HBF is a consultant and advisory board member for Regeneron Pharmaceuticals. IJF is an associate on a cooperative research and development agreement between Merck Pharmaceuticals and the National Institutes for Health. OAH and GDY are Regeneron Pharmaceuticals employees and stockholders and have a pending patent on complement component 5 inhibitor treatment in CHAPLE disease. MJL received support for a federally approved cooperative research and development agreement to support the clinical trial and has a pending patent on complement component 5 inhibitor treatment in CHAPLE disease. All other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

One of the key unit operations during the aseptic fill-finish process of parenteral products, such as biologics, is the filling process of the formulated, sterile filtered drug substance into primary packaging containers. The applied filling technology as well as the process performance majorly impacts final drug product quality. The present review provides an overview of commonly used filling technologies during fill-finish operations of biologics including positive displacement pump systems such as radial peristaltic pump, rotary piston pump, rolling diaphragm pump, or innovative systems such as the linear peristaltic pump, as well as time-over-pressure filling technology. The article describes the operating principle of each pump system and reviews advantages and drawbacks. We highlight specific considerations for individual systems, such as the risk of protein particle formation and particle shedding from wear and tear of tubing, and discuss current literature about general challenges associated with the filling process, such as hydrogen peroxide uptake, adsorption phenomena to tubing material, and needle clogging. We suggest process development and process characterization studies to assess the impact of the filling process on product quality, and lastly provide an outlook about the use of disposable equipment during filling operations related to sustainability considerations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Hapten design and synthesis have been regarded as the key factor to generate high-quality antibodies. In the present study, a novel hapten of chloramphenicol was synthesized, characterized and compared with two conventional haptens. The new hapten generated mAb 4B5 showed higher sensitivity and titer than the other two haptens-based mAbs. The haptens synthesized with the structure of chloramphenicol base generated more sensitive antibodies than the hapten with chloramphenicol succinate, and the spacer arm linked to the phenyl group hapten elicited the strongest antibody response. After optimization, a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA), both based on the mAb 4B5, were developed. The dcELISA had a half maximum inhibition concentration of 0.23 ng/mL and the LFIA showed a cutoff value of 5-10 ng/mL. The LFIA was applied to detect illegally-added chloramphenicol samples in anti-acne cosmetics, five out of 19 samples were tested chloramphenicol containing within 10 min, which result was confirmed with the dcELISA and HPLC. The LFIA has an adequate sensitivity and can be used as a point of care diagnostic device for rapidly screening chloramphenicol in cosmetics., (Copyright © 2023. Published by Elsevier B.V.)

Fenpyroximate is a systemic phenoxy pyrazole acaricide applied worldwide to prevent and control various phytophagous mites and has high activity against young mites, with residues increasingly being found in ecological environment and agricultural products. To identify its residues, four haptens of fenpyroximate were designed and an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on monoclonal antibodies (2G4C7) was developed. Because the icELISA had higher sensitivity to the hydrolysate (Hapten A) of fenpyroximate, a method for indirectly determining the concentration of fenpyroximate was established by measuring the content of Hapten A. The assay had a working range of 0.07-1.49 ng/mL and a half-maximal inhibitory concentration (IC 50 ) of 0.34 ng/mL. It showed average recoveries of 77.0%-105.4 %, 72.3%-106.4 % in citrus and apple samples, respectively. The icELISA and UPLC-MS/MS test results for samples of various citrus cultivars are remarkably consistent. These results and data represent the icELISA is suitable and applicable for detecting fenpyroximate residuals in fruit and vegetable samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Tumor-associated glycoprotein 72 (TAG-72) is a mucin that is overexpressed heterogeneously on the surface of cancer cells, and is a potential target for immunotherapies for various cancer types. As a tumor marker, TAG-72 is measured with the cancer antigen (CA) 72-4 immunoassay. The murine monoclonal antibody (mAb) CC49 is a second-generation IgG that targets an antigen on TAG-72; however, CC49 has an unfavorable propensity to aggregate, which results in antibody impurity, instability, and low solubility and thus low potency and efficacy for therapeutic and diagnostic applications. Sequence analysis of CC49 revealed aggregation-prone motifs in the variable domain of the light chain. Using antibody engineering approaches, we developed three aggregation-resistant CC49 mIgG2a mutants (CC49M1, CC49M2, and CC49M3). The engineered CC49 mIgG2a mutants retained compatible binding performance with a significantly higher thermal stability. The CC49 mIgG2a mutants also demonstrated an almost 15-fold improvement in solubility, with 97% purity vs 70% purity of the parent molecule at 0.3 mg/mL. The enhanced stability and improved solubility of engineered CC49 could have significant advantages for diagnostic and therapeutic applications., Competing Interests: Declaration of Competing Interest The authors are employees of Abbott Laboratories., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Engineered collaborative biochemical techniques and regulated nanomaterials (NMs) offer extraordinary opportunities for improving the analysis performance of lateral flow immunoassay (LFIA). Herein, inspired by the ability of macromolecules (e.g., proteins) to assemble into new functional units and the remarkable optical performance of engineered regulated NMs, goat anti-mouse immunoglobulin (GAMI) serves as the "crosslinker" integrate with gold‑manganese oxide (Au-MnO x ) to assemble the "signal tracers (STs)-crosslinker-antibody (mAb)" for elevating the mAb utilization efficiency. Notably, the "STs-crosslinker-mAb" assembly shows ~13.33-folds mAb utilization efficiency enhance, which perfectly response the challenge between limited sensitivity and sufficient signal intensity in competitive-type LFIA. The black color and rough structure of Au-MnO x offer higher colorimetric brightness (~2-folds than AuNPs) and enhanced mAb coupling efficiency (up to 92.47%), which further improves sensitivity under the premise of functional assembly to intensify the competitive immunoreaction. Additionally, the convenient synthesis conditions (~13 min at room temperature) even comparable to direct purchase commercial products indicate that using Au-MnO x undoubtedly increases the cost-effectiveness. Encouragingly, the Au-MnO x -GAMI-mAb based LFIA exhibited high sensitivity (LOD: 0.063 ng mL -1 for clenbuterol (CLE) monitoring) by elevating mAb utilization efficiency with the attendant enhancing immune competition response in a cost-effective manner, which provides an invigorating reference pathway in point-of-care immunoassay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Therapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone. To improve on this, we developed an integrated and automated multi-dimensional analytical platform for the simultaneous assessment of multiple CQAs of mAbs in cell culture fluid (CCF) from upstream processes., Results: The on-line system allows mAb characterization at the intact level, combining protein A affinity chromatography (ProtA) with size-exclusion, ion-exchange, and reversed-phase liquid chromatographic modes with UV and mass spectrometric detection. Multiple heart cuts of a single mAb elution band from ProtA are stored in 20-μL loops and successively sent to the multimethod options in the second dimension. ProtA loading and elution conditions and their compatibility with second-dimension LC modes were studied and optimized. Subsequently, heart-cutting and valve-switching schemes were investigated to achieve effective and reproducible analyses. The applicability of the developed workflow was demonstrated by the direct analysis (i.e. not requiring off-line sample preparation) of a therapeutic mAb in CCF, obtaining useful information on accurate molecular mass, glycosylation, and charge and size variants of the mAb product at the same time and in just over 1 h., Significance: The developed multidimensional platform is the first system that allows for multiple fractions from a single ProtA band to be characterized using different chromatographic selectivities in a single run allowing direct correlation between CQAs. The performance of the system is comparable to established off-line methods, fully compatible with upstream process samples, and provides a significant time-reduction of the characterization procedure., Competing Interests: Declaration of competing interest The authors declare the following financial interests/ which may be considered as potential competing interests: Govert W. Somsen reports equipment was provided by Agilent Technologies Inc., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

During the manufacturing of therapeutic antibodies, effective Protein A chromatography as initial column step is crucial to simplify the remaining purification effort for subsequent polishing steps. This is particularly relevant for molecules with high impurity content so that desired product purity can be attained. The present study demonstrates beneficial effects on impurity removal when applying kosmotropic salts, e.g., sodium sulfate or sodium chloride, in the elution phase. Initially, a screen using negative linear pH gradient elution evaluated the impact of the kosmotropic salts in comparison to no additive and chaotropic urea using three mAbs and three common resins. Retaining acceptable yield, the kosmotropic salts improved resolution of monomer and impurities and reduced the contents of process-related host cell proteins and DNA as well as of product-related low and high molecular weight forms, despite some resin- and mAb-dependent variations. Moreover, a decrease in hydrolytic activity measured by a new assay for polysorbase activity was observed. In contrast, urea was hardly effective. The findings served to establish optimized step elution conditions with 0.25 M of sodium sulfate for a challenging mAb with complex format (bispecific 2 + 1 CrossMab) displaying high relative hydrophobicity and impurity levels. With yield and purity both in the range of 90 %, the contents of all impurity components were reduced, e.g., low molecular weight forms by two-fold and polysorbase activity by four-fold. The study indicates the potential of kosmotropic salts to establish efficient and comprehensive impurity separation by Protein A for facilitated downstream processing and economic manufacturing of complex antibodies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Monoclonal antibody downstream processing typically entails chromatography-based purification processes beginning with Protein A chromatography, accounting for 50 % of the total manufacturing expense. Alternatives to protein A chromatography have been explored by several researchers. In this paper, aqueous two-phase extraction (ATPE) has been proposed for continuous processing of monoclonal antibodies (mAbs) as an alternative to the traditional protein A chromatography. The PEG-sulfate system has been employed for phase formation in ATPE, and the mAb is separated in the salt phase, while impurities like high molecular weight (HMW) and host cell proteins (HCPs) are separated in the PEG phase. Following ATPE of clarified cell culture harvest, yield of ≥ 80 % and purity of ≥ 97 % were achieved in the salt phase. Considerable (28 %) reduction in consumable cost has been estimated when comparing the proposed platform to the traditional protein A based platform. The outcomes demonstrate that ATPE can be a potentially effective substitute for the traditional Protein A chromatography for purification of mAbs. The proposed platform offers easy implementation, delivers comparative results, and offers significantly better economics for manufacturing mAb-based biotherapeutics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Background: Fibrinogen γ' is a naturally occurring 20-amino-acid splice variant of the fibrinogen γ chain. Animal studies link variations in fibrinogen to obesity, but it is unknown how fibrinogen γ' is associated with obesity in humans., Objectives: To develop and validate an enzyme-linked immunosorbent assay (ELISA) for fibrinogen γ' quantification in human plasma and analyze fibrinogen γ' before and after bariatric surgery., Methods: We generated C-terminal fibrinogen γ' specific mouse monoclonal antibodies and developed a γ' ELISA. Validation included measures of accuracy, sensitivity, and precision. Fibrinogen γ' and total fibrinogen were measured in 60 individuals before and 6 months after bariatric surgery and in 19 normal-weight controls and 120 blood donors., Results: Highly specific fibrinogen γ' monoclonal antibodies were produced and successfully used in the ELISA. Recovery was 88%, and limits of detection and quantification were 0.003 mg/mL and 0.014 mg/mL, respectively. Coefficients of variation were 3% for repeatability and 7% for within-laboratory variation. The fibrinogen γ' reference interval was 0.25 to 0.80 mg/mL. Fibrinogen γ' concentrations were reduced after bariatric surgery and were higher in individuals with obesity than in those with normal weight. The fibrinogen γ'/total fibrinogen ratio was unchanged after surgery but was higher than the ratio in normal-weight individuals., Conclusion: We developed a precise and sensitive ELISA for fibrinogen γ'. Levels of fibrinogen γ', but not the fibrinogen γ'/fibrinogen ratio, were reduced 6 months after bariatric surgery. Absolute and relative levels of fibrinogen γ' were increased in individuals with obesity compared to normal-weight individuals., (© 2024 The Authors.)

Monoclonal antibodies are utilized for treating many diseases and disorders, as well as for basic research and development. Covalent labeling of mAbs is important for various antibody applications and creating antibody drug conjugates. Labeling at reactive lysine residues using lysine selective reagents is useful, but is non-selective and can interfere with antigen binding and interactions of the Fc antibody region. In this work, using an anti-cocaine mAb (h2E2), we utilized triphenylphosphine-3,3',3″-trisulfonic acid (TPPTS), and demonstrated for the first time reduction of disulfides in an antibody by TPPTS. More importantly, this reduction was very reproducible, limited, and selective, and permitted selective labeling of the antibody with a cysteine reactive fluorescent reagent, resulting in labeling of a few specific cysteines. Similar results were obtained using TCEP-agarose reduction. We demonstrated that both of these selective partial reduction methods gave rise to approximately two labels per mAb, mostly by selective reduction of the heavy chain to light chain disulfide bond, as demonstrated by non-reducing SDS-PAGE protein band analysis. Thus, convenient, reproducible, and selective mAb disulfide reduction was achieved under mild conditions. These labeled, partially reduced mAbs were characterized by differential scanning fluorimetry (DSF), detecting the incorporated fluorescein instead of an exogenously added dye, and for antigen (cocaine) binding by isothermal titration calorimetry (ITC). Both the structure and antigen binding of the mAb was maintained. This novel selective reduction and labeling is generally relevant to modification of antibodies and to future development of conjugated mAbs for experimental and therapeutic purposes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Andrew B. Norman has patent #10501556 issued to University of Cincinnati, E. R. Squibb & Sons, L.L.C. Andrew B. Norman has patent #9957332 issued to University of Cincinnati. Andrew B. Norman has patent #9758593 issued to University of Cincinnati, E. R. Squibb & Sons, L.L.C., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Alternariol is a toxic secondary metabolite produced by Alternaria fungi. Nowadays, this mycotoxin can be found in many products of plant origin at concerning concentrations. The aim of the present study was to develop a highly sensitive and selective immunochemical method for the analysis of alternariol. To this end, hapten synthesis was carried out from the scratch and specific high-affinity monoclonal antibodies to alternariol were generated by using, for the first time, alternariol bioconjugates with unambiguous linker tethering sites. A novel indirect enzyme-linked immunosorbent assay that incorporated a rationally designed heterologous conjugate was developed. The optimized assay showed an outstanding half-maximal inhibition concentration for alternariol below 0.04 ng/mL, and no cross-reactivity with alternariol monomethyl ether was observed. Very good recovery values and coefficients of variation were obtained from the analysis of alternariol-fortified fruit and cereal flour samples. Finally, alternariol was quantitatively determined in pears that had been previously infected with Alternaria alternata, showing excellent correlation with liquid chromatography results., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Sulfamethazine (SM2) is an antibacterial drug，which has been extensively used in human and veterinary medicine, long-term consumption of which may lead to the accumulation of sulfonamides in the body. Detection of sulfonamides often uses microbiological approaches, mass spectrometry and chromatography, which are expensive and time-consuming. Surface-enhanced Raman scattering-based immunochromatographic assay (SERS-ICA) has been recently applied in the detection. Herein, a Janus-labeled Au nanoparticle with subnanosized SiO 2 -monoclonal antibody and SERS reporter (DTNB) modified simultaneously ( mAb AuNp DTNB ) has been developed in a SERS-based lateral flow immunosensor, which can be used for rapid, quantitative and ultrasensitive detection of sulfamethazine residue in milk. The mAb AuNp DTNB exhibits a specific array on a paper stripe, which not only identifies sulfamethazine but also straightforwardly exposes the Raman reporter between the AuNps via self-assembly. The detection sensitivity of SERS-ICA for sulfamethazine reached 0.1 pg/mL, which was far below the previously published value by ELISA and the maximum residue limit set by the European Union. The entire SERS-ICA detection for sulfamethazine was completed within 15 min. Furthermore, high accuracy for this assay was exhibited in the spiking experiment with a recovery percentage of 88.1%-112.7%. The results demonstrated that this SERS-ICA can potentially be applied in point-of-care testing as an ultrasensitive and quantitative to semi-quantitative analytical method., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023. Published by Elsevier B.V.)

The evidence base for migraine prevention in both episodic and chronic migraine is outlined. The older oral preventatives, including antidepressants, antihypertensives, serotonin antagonists, antiepileptics, and calcium channel antagonists, and newer options including onabotulinumtoxinA and the CGRP monoclonal antibodies are covered. Many of the older oral preventatives were trialed before chronic migraine was defined, and they are used in chronic migraine based on the assumption that episodic migraine and chronic migraine are on a spectrum of the same condition. First- and second-line options are given, and a multicountry perspective is considered., (Copyright © 2024 Elsevier B.V. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Colorectal cancer (CRC) is a common malignant tumor of the gastrointestinal tract and one of the most common causes of cancer-related deaths worldwide. Immune checkpoint inhibitors have become a milestone in many cancer treatments with significant curative effects. However, its therapeutic effect on colorectal cancer is still limited. B7-H3 is a novel immune checkpoint molecule of the B7/CD28 family and is overexpressed in a variety of solid tumors including colorectal cancer. B7-H3 was considered as a costimulatory molecule that promotes anti-tumor immunity. However, more and more studies support that B7-H3 is a co-inhibitory molecule and plays an important immunosuppressive role in colorectal cancer. Meanwhile, B7-H3 promoted metabolic reprogramming, invasion and metastasis, and chemoresistance in colorectal cancer. Therapies targeting B7-H3, including monoclonal antibodies, antibody drug conjugations, and chimeric antigen receptor T cells, have great potential to improve the prognosis of colorectal cancer patients., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest with respect to the research, author-ship, and publication of this article., (Copyright © 2023. Published by Elsevier B.V.)

To determine the disease status and the response to treatment for patients with multiple myeloma, measuring serum M-protein levels is a widely used alternative to invasive punctures to count malignant plasma cells in the bone marrow. However, the quantification of this monoclonal antibody, which varies from patient to patient, poses significant analytical challenges. This paper describes a sensitive and specific mass spectrometry assay that addresses two objectives: to overcome the potential interference of biotherapeutics in the measurement of M-proteins, and to determine the depth of response to treatment by assessing minimal residual disease. After immunocapture of immunoglobulins and free light chains in serum, heavy and light chains were dissociated by chemical reduction and separated by liquid chromatography. M-proteins were analyzed by high-resolution mass spectrometry using a method combining a full MS scan for isotyping and identification and a targeted single ion monitoring scan for quantification. This method was able to discriminate M-protein from the therapeutic antibody in all patient samples analyzed and allowed quantification of M-protein with a LLOQ of 2.0 to 3.5 µg/ml in 5 out of 6 patients. This methodology appears to be promising for assessing minimal residual disease with sufficient sensitivity, specificity, and throughput., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)