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Apolipoprotein AV (APOA5) deficiency causes hypertriglyceridemia in mice and humans. For years, the cause remained a mystery, but the mechanisms have now come into focus. Here, we review progress in defining APOA5s function in plasma triglyceride metabolism. Biochemical studies revealed that APOA5 binds to the angiopoietin-like protein 3/8 complex (ANGPTL3/8) and suppresses its ability to inhibit the activity of lipoprotein lipase (LPL). Thus, APOA5 deficiency is accompanied by increased ANGPTL3/8 activity and lower levels of LPL activity. APOA5 deficiency also reduces amounts of LPL in capillaries of oxidative tissues (e.g., heart, brown adipose tissue). Cell culture experiments revealed the likely explanation: ANGPTL3/8 detaches LPL from its binding sites on the surface of cells, and that effect is blocked by APOA5. Both the low intracapillary LPL levels and the high plasma triglyceride levels in Apoa5-/- mice are normalized by recombinant APOA5. Carboxyl-terminal sequences in APOA5 are crucial for its function; a mutant APOA5 lacking 40-carboxyl-terminal residues cannot bind to ANGPTL3/8 and lacks the ability to change intracapillary LPL levels or plasma triglyceride levels in Apoa5-/- mice. Also, an antibody against the last 26 amino acids of APOA5 reduces intracapillary LPL levels and increases plasma triglyceride levels in wild-type mice. An inhibitory ANGPTL3/8-specific antibody functions as an APOA5-mimetic reagent, increasing intracapillary LPL levels and lowering plasma triglyceride levels in both Apoa5-/- and wild-type mice. That antibody is a potentially attractive strategy for treating elevated plasma lipid levels in human patients.

[This corrects the article DOI: 10.3389/fimmu.2023.1207108.].

The optimal efficacy of xenogeneically generated proteins intended for application in humans requires that their own antigenicity be minimized. This necessary adaptation of antibodies to a humanized version poses challenges since modifications even distant from the binding sites can greatly influence antigen recognition and this is the primary feature that must be maintained during all modifications. Current strategies often rely on grafting and/or randomization/selection to arrive at a humanized variant retaining the binding properties of the original molecule. However, in terms of speed and efficiency, rationally directed approaches can be superior, provided the requisite structural information is available. We present here a humanization procedure based on the high-resolution X-ray structure of a chimaeric IgG against a marker for multiple myeloma. Based on in silico modelling of humanizing amino acid substitutions identified from sequence alignments, we devised a straightforward cloning procedure to rapidly evaluate the proposed sequence changes. Careful inspection of the structure allowed the identification of a potentially problematic amino acid change that indeed disrupted antigen binding. Subsequent optimization of the antigen binding loop sequences resulted in substantial recovery of binding affinity lost in the completely humanized antibody. X-ray structures of the humanized and optimized variants demonstrate that the antigen binding mode is preserved, with surprisingly few direct contacts to antibody atoms. These results underline the importance of structural information for the efficient optimization of protein therapeutics. Key messages: Structure-based humanization of an IgG against BCMA, a marker for Multiple Myeloma. Identification of problematic mutations and unexpected modification sites. Structures of the modified IgG-antigen complexes verified predictions. Provision of humanized high-affinity IgGs against BCMA for therapeutic applications. [ABSTRACT FROM AUTHOR]

Background: Numerous studies suggest a progressive accumulation of post-translationally modified peptides within amyloid fibrils, including isoaspartate (isoD) modifications. Here, we generated and characterised novel monoclonal antibodies targeting isoD-modified transthyretin (TTR). The antibodies were used to investigate the presence of isoD-modified TTR in deposits from transthyretin amyloidosis patients and to mediate antibody-dependent phagocytosis of TTR fibrils. Methods: Monoclonal antibodies were generated by immunisation of mice using an isoD-modified peptide and subsequent hybridoma generation. The antibodies were characterised in terms of affinity and specificity to isoD-modified TTR using surface plasmon resonance, transmission electron microscopy and immunohistochemical staining of human cardiac tissue. The potential to elicit antibody-dependent phagocytosis of TTR fibrils was assessed using THP-1 cells. Results: We developed two mouse monoclonal antibodies, 2F2 and 4D4, with high nanomolar affinity for isoD-modified TTR and strong selectivity over the unmodified epitope. Both antibodies show presence of isoD-modified TTR in human cardiac tissue, but not in freshly purified recombinant TTR, suggesting isoD modification only present in aged fibrillar deposits. Likewise, the antibodies only facilitated phagocytosis of TTR fibrils and not TTR monomers by THP-1 cells. Conclusions: These antibodies label aged, non-native TTR deposits, leaving native TTR unattended and thereby potentially enabling new therapeutic approaches. [ABSTRACT FROM AUTHOR]

RSV bronchiolitis remains the leading cause of hospitalization in children under 1 year of age. It is estimated that 2–6% of cases will be hospitalized on pediatric intensive care units (PICUs). In October 2023, a universal immunization program with the monoclonal antibody nirsevimab was implemented in Catalonia. The aim of the study was to analyze the impact of the nirsevimab immunization on the burden of bronchiolitis admitted to a PICU and resulting changes in epidemiological, clinical, and microbiological characteristics comparing the pre-nirsevimab (pre-N) with the post-nirsevimab (post-N) period. This was a prospective, descriptive, and observational study. Patients with severe bronchiolitis admitted to reference children's hospital PICU, between September 2010 and February 2024 were included. Demographic and clinical data were collected and viral laboratory etiological diagnosis was carried out. 1531 patients were recruited, 1458 in the pre-N seasons and 73 after its introduction (58% males, median age 52 days), of which 67% were immunized with nirsevimab. The total number of PICU bronchiolitis admissions, the ratio, and the RSV etiology were significantly lower in the post-N period (p = 0.03, p < 0.001, and p = 0.039, respectively). Significant higher age at admission (p < 0.001) and lower hospital length of stay (p < 0.001) was observed comparing pre-N vs. post-N period. Conclusion: Nirsevimab appears to have an important impact on reducing the number and length of stay of PICU admissions due to RSV bronchiolitis. What Is Known: • Bronchiolitis is the most common viral infection of the lower respiratory tract in infants. • It represents 13% of the total pediatric intensive care admissions, typically during winter. This is one of the causes that produces a collapse in the health care systems all around the world. What Is New: • In October 2023, universal immunization with monoclonal antibody nirsevimab of all children under 6 months of age was started in the majority of autonomous communities in Spain. • Recent publications from the nirsevimab clinical trials have evidenced a high RSV protective effect, but data on its effect on real life patients who require pediatric intensive care unit admission are missing. [ABSTRACT FROM AUTHOR]

Herbicide residues in agricultural products can have adverse effects on the environment and human health, therefore, there is an urgent need to establish a sensitive, rapid, and wide-ranging detection method. In this study, haptens of phenylurea herbicides (PUs) and sulfonylurea herbicides (SUs) were analyzed and designed based on computational simulation techniques, and two high-performance broad-spectrum monoclonal antibodies against PUs and SUs were prepared. On this basis, a multi-colloidal gold immunochromatography assay (multi-CGIA) was developed to simultaneously detect 13 herbicides in wheat. The visual limit of detection (vLOD) for PUs including diuron, chlortoluron, neburon, chlorbromuron, and linuron was 1–2 µg/kg. The vLOD for SUs including metsulfuron methyl, ethametsulfuron-methyl, sulfometuron-methyl, tribenuron methyl, cinosulfuron, triasulfuron, chlorimuron-ethyl, and chlorsulfuron was 2–10 µg/kg. The results of real sample determination indicated that the multi-CGIA is accurate, stable, and reliable, and adaptable to on-site preliminary screening of actual samples. [ABSTRACT FROM AUTHOR]

Poly(butyl methacrylate‐co‐ethylene dimethacrylate) monolith was in situ prepared in a liquid chromatography capillary column with a 75 μm internal diameter. This monolith offered high permeability (5.3 ± 10−14 m2) and good peak capacity (140 for a 15 cm column length at 300 nl/min with a 20 min gradient time). This is exemplified by its separation ability in reversed mode for subunit analysis of monoclonal antibodies after IdeS digestion (middle‐up analysis). The potential of this column was also illustrated for the fast analytical control of therapeutic monoclonal antibodies in standardized infusion bags prepared in advance in a pharmacy department. Linearity analysis revealed the column's capability for accurate quantification analysis of the different dose bandings (in mg) of monoclonal antibodies in <2 min. In addition, lifetime analysis data indicated that the column can be highly reproducible and has a long lifetime with stable and low back pressure. The variations observed on the peak shape and area between unstressed (intact) and stressed monoclonal antibodies indicated that our nano liquid chromatographic method was stability indicating. In addition, using a gradient elution mode, the presence of minor components in the infusion bags was visualized. [ABSTRACT FROM AUTHOR]

Entamoeba nuttalli is genetically the closest to Entamoeba histolytica, the causative agent of human amebiasis. E. nuttalli is found in Macaca species, exhibiting no symptoms while potentially virulent. Using comparative genomics of Entamoeba species, we identified a gene encoding an E. nuttalli‐specific protein containing 42 repeats of an octapeptide (PTORS). In the present study, we analyzed the genes in E. nuttalli strains derived from various geographic locations and host species. Sequence analysis of genomic DNA from four strains indicated 43, 44, and 48 repeat types in addition to 42 repeats and remarkable genetic diversity in the repeat region, although all nucleotide substitutions were synonymous. In contrast, the sequences of the N‐terminal side region and C‐terminus were identical among the strains. Monoclonal antibodies prepared against recombinant PTORS were reactive to the repeat regions but not to the N‐terminal side regions. Polyclonal antibodies did not react with the N‐terminal region, demonstrating that the repeat region had higher antigenicity. Analysis using synthetic peptides revealed that the two repeats of the octapeptide functioned as epitopes. Immunofluorescence microscopy using monoclonal antibodies demonstrated the surface localization of PTORS. These results suggest that the repeat region of PTORS plays an important role in host–parasite interactions. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) is a highly fatal infectious disease in pigs, caused by the African swine fever virus (ASFV). It is characterized by short disease duration and high morbidity and mortality. In August 2018, ASF was first reported in China and it subsequently spread rapidly throughout the country, causing serious economic losses for the Chinese pig industry. Early detection plays a critical role in preventing and controlling ASF because there is currently no effective vaccine or targeted therapeutic medication available. Additionally, identifying conserved protective antigenic epitopes of ASFV is essential for the development of diagnostic reagents. The E165R protein, which is highly expressed in the early stages of ASFV infection, can serve as an important indicator for early detection. In this study, we successfully obtained high purity soluble prokaryotic expression of the E165R protein. We then utilized the purified recombinant E165R protein for immunization in mice to prepare monoclonal antibodies (mAbs) using the hybridoma fusion technique. After three subclonal screens, we successfully obtained three mAbs against ASFV E165R protein in cells named 1B7, 1B8, and 10B8. Through immunofluorescence assay (IFA) and Western blot, we confirmed that the prepared mAbs specifically recognize the baculovirus-expressed E165R protein. By using overlapping truncated E165R protein and overlapping peptide scanning analysis, we tentatively identified two novel linear B cell epitopes (13EAEAYYPPSV22 and 55VACEHMGKKC64) that are highly conserved in genotype I and genotype II of ASFV. Thus, as a detection antibody, it has the capability to detect ASFV across a wide range of genotypes, providing valuable information for the development of related immunodiagnostic reagents. [ABSTRACT FROM AUTHOR]

AbstractPseudomonas cocovenenans subsp. farinofermentans is a pathogenic bacterium that causes the poisoning of fermented rice products. Its isomeric products, bongkrekic acid (BA) and isobongkrekic acid (IBA), have become popular research topics. In this study, we developed a heterologous-coating strategy for high-sensitivity indirect competitive enzyme-linked immunoassay (icELISA) for BA and IBA using monoclonal antibodies (mAbs). The optimized procedure exhibited an IC50 of 5.49 ng/mL, a limit of detection of 0.99 ng/mL, and a linear range from 2.12 to 14.21 ng/mL. Notably, this method exhibited negligible cross-reactivity to other small toxins, and the recoveries of the samples, including rice noodles, Auricularia auricula, and flat rice noodles, ranged from 92.0%–111.9%, 105.6%–115.0%, and 83.2%–109.0%, respectively, with coefficients of variation below 12%. A comparison of the recoveries of high-performance liquid chromatography-mass spectrometry (HPLC-MS) and icELISA demonstrated that the developed approach provides good accuracy and reliability. Overall, the simple preparation of IBA combined with the suitability of icELISA for high-throughput analysis is efficient and cost-effective, making it suitable for routine screening of food. [ABSTRACT FROM AUTHOR]

Background Objectives Animals Materials and Methods Results Conclusion and Clinical Relevance Lokivetmab, a caninised monoclonal antibody against interleukin (IL)‐31, is an effective treatment for the pruritus associated with canine atopic dermatitis (cAD).To investigate the efficacy and safety of lokivetmab during long‐term treatment defined as at least three consecutive lokivetmab injections in atopic dogs under field conditions. To assess individual factors influencing treatment outcome and adverse events.150 dogs with cAD.Medical records of dogs treated with lokivetmab were reviewed, and owners and/or veterinarians were contacted as needed for follow‐up. A decrease of the pruritus Visual Analog Scale (PVAS) score by ≥2 or a PVAS score ≤2 after treatment was considered as treatment success. Logistic regression was used to investigate the influence of a variety of factors on outcome: type of cAD (food versus environment), age at first lokivetmab administration, disease chronicity, dosage and/or secondary infection. Any adverse event that occurred during the study period was recorded.Lokivetmab reduced the PVAS score with long‐term use (p < 0.01); the success rate was 53 of 69 total dogs (77%). The probability of treatment failure decreased with increasing treatment duration. None of the factors investigated influenced the treatment outcome. Twelve dogs of 150 (8%) showed adverse events such as gastrointestinal signs or lethargy.Lokivetmab appears to be an effective and safe long‐term anti‐itch therapy for dogs with cAD. [ABSTRACT FROM AUTHOR]

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Exidavnemab is a monoclonal antibody (mAb) with a high affinity and selectivity for pathological aggregated forms of α‐synuclein and a low affinity for physiological monomers, which is in clinical development as a disease‐modifying treatment for patients with synucleinopathies such as Parkinson's disease. Safety, tolerability, pharmacokinetics, immunogenicity, and exploratory biomarkers were assessed in two separate Phase 1 single ascending dose studies, including single intravenous (IV) (100 to 6000 mg) or subcutaneous (SC) (300 mg) administration of exidavnemab in healthy volunteers (HVs). Across the two studies, a total of 98 Western, Caucasian, Japanese, and Han Chinese HVs were enrolled, of which 95 completed the study. Exidavnemab was generally well tolerated. There were no serious adverse events or safety issues identified in laboratory analyses. Headache, asymptomatic COVID‐19, back pain, and post lumbar puncture syndrome were the most frequently reported treatment‐emergent adverse events. Following IV infusion, the pharmacokinetics of exidavnemab was approximately dose linear in the range 100‐6000 mg. The terminal half‐life was approximately 30 days, and the exposure was comparable across Western, Caucasian, Japanese, and Han Chinese volunteers. The absolute SC bioavailability was ∼71%. Cerebrospinal fluid exposure relative to serum after single dose was within the range expected for mAbs (approximately 0.2%). The anti‐drug antibody rates were low and there was no effect of immunogenicity on the pharmacokinetics or safety. Dose‐dependent reduction of free α‐synuclein in plasma was observed. In summary, exidavnemab was found to have an excellent pharmacokinetic profile and was well tolerated in HVs, supporting the continued clinical development. [ABSTRACT FROM AUTHOR]

Single B-cell antibody generation technology is an advanced method that offers several advantages, including rapid production, high efficiency, and high yield. The antibodies generated via this technique retain their natural conformation and are well suited for applications in pathogen diagnosis, disease treatment, and investigations of virus cross-species transmission mechanisms. Our study aimed to establish a platform for generating single B-cell antibodies specifically targeting the foot-and-mouth disease virus (FMDV) 146S antigen in mice. Female BALB/c mice were immunized with inactivated O-type FMDV 146S antigen, and spleen cells were collected for further analysis. Flow cytometric sorting was performed using a biotin-labeled O-type FMDV 146S antigen as a decoy to identify and select CD19 + /CD21/35 + /CD43-/IgM-/Biotin + antigen-specific individual B cells. The gene sequences encoding the variable regions of the heavy and light chains of the murine IgG antibodies were obtained via single-cell nested PCR amplification. Separate constructs were created for the heavy and light chain plasmids to ensure the proper expression of intact IgG antibodies. These plasmids were cotransfected into human embryonic kidney 293T (HEK293T) cells, leading to the successful production and purification of 15 specific monoclonal antibodies (mAbs), 10 which exhibited activity in ELISA tests, and six antibodies that displayed activity in IFA tests. These findings highlight the successful development of a method for generating mouse-derived single B-cell antibodies that target FMDV. This achievement provides a solid foundation for diagnostic techniques and the analysis of antigenic structural variations. [ABSTRACT FROM AUTHOR]

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the F ab domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the C H 2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone. [ABSTRACT FROM AUTHOR]

Thermal stability attributes including unfolding onset (T onset) and mid-point (T m) are often utilized for efficient development of monoclonal antibody (mAb) products during lead selection and formulation screening workflows. An assumption of direct correlation between thermal and kinetic physical stability underpins this basic approach. While literature reports have substantiated this general approach under specific conditions, clear exceptions have been highlighted alongside. Herein, a set of mAbs formulated under diverse solution conditions to generate a broad array of thermal and kinetic stability profiles were systematically analyzed. Sequence modifications in the Fc region were purposefully engineered to generate a set of low-melting mAbs. A diverse set of excipients were subsequently utilized and shown to modulate the T m over a wide range. While a general correlation between high T m and low aggregation rate was observed under accelerated conditions, the predictive utility of T m under relevant product storage conditions was inadequate at best. Critically, T m data did not correlate with long-term aggregation rates under refrigerated or room temperature conditions. Even under accelerated conditions, T m appeared to be a poor predictor of aggregation once it exceeded the solution storage temperature (40°C) by ∼15°C, similar to conditions routinely encountered in the development of canonical mAbs (T m > 60°C). Pitfalls of simplistic correlative approaches are discussed in the context of practical biologics product development. [ABSTRACT FROM AUTHOR]

Sudan ebolavirus (SUDV) is a member of the genus Ebolavirus (Family Filoviridae) and has caused sporadic outbreaks of Ebola disease (EBOD), or more specifically Sudan virus disease (SVD), with high mortality rates in Africa. Current vaccines and therapies that have been developed for filoviruses are almost all specific for Ebola virus (EBOV; of the species Zaire ebolavirus), and there is a current lack of therapeutics specific for SUDV. The recent SUDV outbreak in Uganda, which was distributed across multiple districts, including Kampala, a densely populated urban center, highlights the critical need for the development of novel SUDV-specific or pan-Ebola virus therapeutics. Previous work has characterized two monoclonal antibodies, FVM04 and CA45, which have neutralization capabilities against both EBOV and SUDV and have shown protective efficacy in animal challenge studies. Here, we expand upon this work, showing that treatment with a monoclonal antibody cocktail consisting of FVM04 and CA45 provides full protection against lethal SUDV infection in cynomolgus macaques. Studies that evaluate outcomes at late time points after infection, once clinical signs of illness are apparent, are vital for assessing the therapeutic efficacy of antibody therapeutics. We have shown that when treatment is initiated as late as 5 days after infection, with a second dose given on day 8, that treated groups showed few clinical signs or morbidity, with complete survival. This work provides further evidence that FVM04 and CA45 have strong therapeutic potential against SUDV and their development as a pan-Ebola virus therapeutic should be pursued. IMPORTANCE There are currently no approved vaccines or therapeutics for Sudan virus, a filovirus which is highly related to Ebola virus and causes similar disease and outbreaks. In this study, a cocktail of two potent monoclonal antibodies that effectively neutralize Sudan virus was tested in a nonhuman primate model of Sudan virus disease. Treatment was highly effective, even when initiated as late as 5 days after infection, when clinical signs of infection were already evident. All treated animals showed complete recovery from infection, with little evidence of disease, while all animals that received a control treatment succumbed to infection within 8 days. The study further demonstrated the strong therapeutic potential of the antibody treatment and supported further development for use in Sudan virus outbreaks. [ABSTRACT FROM AUTHOR]

One of the G protein-coupled receptors, C-C chemokine receptor 5 (CCR5), is an important regulator for the activation of T and B lymphocytes, dendritic cells, natural killer cells, and macrophages. Upon binding to its ligands, CCR5 activates downstream signaling, which is an important regulator in the innate and adaptive immune response through the promotion of lymphocyte migration and the secretion of proinflammatory cytokines. Anti-CCR5 monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials for tumors and inflammatory diseases. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the N-terminal peptide immunization. Among the established anti-mCCR5 mAbs, C5Mab-4 (rat IgG2a, kappa) and C5Mab-8 (rat IgG1, kappa), recognized mCCR5-overexpressing Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. The dissociation constant (KD) values of C5Mab-4 and C5Mab-8 for CHO/mCCR5 were determined as 3.5 × 10−8 M and 7.3 × 10−9 M, respectively. Furthermore, both C5Mab-4 and C5Mab-8 could detect mCCR5 by western blotting. These results indicated that C5Mab-4 and C5Mab-8 are useful for detecting mCCR5 by flow cytometry and western blotting and provide a possibility to obtain the proof of concept in preclinical studies. [ABSTRACT FROM AUTHOR]

C-C chemokine receptor 5 (CCR5), a member of the G protein-coupled receptor family, is the most common coreceptor for the human immunodeficiency virus type 1. CCR5 is also involved in the pathogenesis of tumors and inflammatory diseases. The CCR5 antagonists including monoclonal antibodies (mAbs) have been developed and evaluated in clinical trials. In this study, we developed novel mAbs for mouse CCR5 (mCCR5) using the Cell-Based Immunization and Screening (CBIS) method. One of the established anti-mCCR5 mAbs, C5Mab-2 (rat IgG2b, kappa), reacted with mCCR5-overexpressed Chinese hamster ovary-K1 (CHO/mCCR5) and an endogenously mCCR5-expressing cell line (L1210) by flow cytometry. Using flow cytometry, the dissociation constant (KD) of C5Mab-2 for CHO/mCCR5 was determined as 4.3 × 10−8 M. These results indicated that C5Mab-2 is useful for the detection of mCCR5 in flow cytometry and may be applicable to obtain the proof of concept in preclinical studies. [ABSTRACT FROM AUTHOR]

The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8+ cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C8Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C8Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C8Mab-2 recognizes the N-terminal region (1–33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17-DFFTAP-22 sequence is important for the recognition by C8Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C8Mab-2. [ABSTRACT FROM AUTHOR]

Phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is a de novo purine biosynthetic enzyme. It has been found to be overexpressed in various types of cancer and is related to cell proliferation, invasion, the epithelial–mesenchymal transition, and efficient tumor growth. In this study, we describe a rat monoclonal antibody (mAb) 6A10, which was generated as an antigen of human PAICS. This mAb was generated to interact with the N-terminal region of human PAICS and was found to recognize endogenous PAICS enzymes in several cancer cells. Our results also indicated that it can recognize monkey and dog PAICS, which possess the same amino acid sequence in the antigenic region as human PAICS, but it does not recognize rat and mouse PAICS. Furthermore, our data indicated that this mAb is suitable for immunoprecipitation and immunoblotting use for several cancer cell lines. We, therefore, anticipate that mAb 6A10 will be useful for functional analyses of human PAICS in several cancers and for diagnosis of malignant transformation. [ABSTRACT FROM AUTHOR]

Interstitial cystitis/bladder pain Syndrome (IC/BPS) remains a mysterious and intricate urological disorder, presenting significant challenges to healthcare providers. Traditional guidelines for IC/BPS follow a hierarchical model based on symptom severity, advocating for conservative interventions as the initial step, followed by oral pharmacotherapy, intravesical treatments, and, in refractory cases, invasive surgical procedures. This approach embraces a multi-tiered strategy. However, the evolving understanding that IC/BPS represents a paroxysmal chronic pain syndrome, often involving extravesical manifestations and different subtypes, calls for a departure from this uniform approach. This review provides insights into recent advancements in experimental strategies in animal models and human studies. The identified therapeutic approaches fall into four categories: (i) anti-inflammation and anti-angiogenesis using monoclonal antibodies or immune modulation, (ii) regenerative medicine, including stem cell therapy, platelet-rich plasma, and low-intensity extracorporeal shock wave therapy, (iii) drug delivery systems leveraging nanotechnology, and (iv) drug delivery systems assisted by energy devices. Future investigations will require a broader range of animal models, studies on human bladder tissues, and well-designed clinical trials to establish the efficacy and safety of these therapeutic interventions. [ABSTRACT FROM AUTHOR]

Acute myocardial infarction is an important cardiovascular disease worldwide. Although the mortality rate of myocardial infarction (MI) has improved dramatically in recent years due to timely treatment, adverse remodeling of the left ventricle continues to affect cardiac function. Various immune cells are involved in this process to induce inflammation and amplification. The infiltration of inflammatory cells in the infarcted myocardium is induced by various cytokines and chemokines, and the recruitment of leukocytes further amplifies the inflammatory response. As an increasing number of clinical anti-inflammatory therapies have achieved significant success in recent years, treating myocardial infarction by targeting inflammation may become a novel therapeutic option. In particular, successful clinical trials of canakinumab have demonstrated the important role of the inflammatory factor interleukin-1 (IL-1) in atherosclerosis. Targeted IL-1 therapy may decrease inflammation levels and improve cardiac function in patients after myocardial infarction. This article reviews the complex series of responses after myocardial infarction, including the involvement of inflammatory cells and the role of cytokines and chemokines, focusing on the progression of the IL-1 family in myocardial infarction as well as the performance of current targeted therapy drugs in experiments. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus Protein A was immobilized on an in‐house made neutravidin poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) capillary column with a 25 µm internal diameter, a length of 30 mm and a mass loadability of 60 ng. The immobilized quantity of protein A on the organic monolith was very low, in the pico mole range (1.80 pmol). This was of significance importance when working with less available or expensive purified enzyme. This capillary column was integrated into a nano liquid chromatographic system and used for the fast determination without dilution of the doses of therapeutic monoclonal antibodies (mAbs) in standardized infusion bags prepared in advance in a pharmacy department. This chromatographic method was linear in the studied concentration range with good precision and accuracy. Heat stressed studies indicated that the protein A affinity capillary column was able to monitor degraded mAbs. As well, the high specificity of this column to capture immunoglobulin G2 in cell culture supernatant was visualized. As the mAbs are produced through genetic engineering of animal cells this last result demonstrated that this novel protein A column could be used in the feature for rapid screening of immunoglobulin G concentration in cell culture. [ABSTRACT FROM AUTHOR]

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder resulting from the deposition of misfolded and neurotoxic forms of tau protein in specific areas of the midbrain, basal ganglia, and cortex. It is one of the most representative forms of tauopathy. PSP presents in several different phenotypic variations and is often accompanied by the development of concurrent neurodegenerative disorders. PSP is universally fatal, and effective disease-modifying therapies for PSP have not yet been identified. Several tau-targeting treatment modalities, including vaccines, monoclonal antibodies, and microtubule-stabilizing agents, have been investigated and have had no efficacy. The need to treat PSP and other tauopathies is critical, and many clinical trials investigating tau-targeted treatments are underway. In this review, the PubMed database was queried to collect information about preclinical and clinical research on PSP treatment. Additionally, the US National Library of Medicine's ClinicalTrials.gov website was queried to identify past and ongoing clinical trials relevant to PSP treatment. This narrative review summarizes our findings regarding these reports, which include potential disease-modifying drug trials, modifiable risk factor management, and symptom treatments. [ABSTRACT FROM AUTHOR]

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC–MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC–MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC–MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC–MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC–MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals. [ABSTRACT FROM AUTHOR]

This review article will summarize the vaccines and monoclonal antibodies currently under evaluation for the prevention of RSV disease in older infants, toddlers and young children. We will review the rationale for passive protection during the first months of life, and the role of active immunization afterwards, either with live attenuated, protein-based or mRNA vaccines. [ABSTRACT FROM AUTHOR]

To date, safe and effective strategies to prevent medically attended respiratory syncytial virus (RSV) illness across the infant population have been limited to passive immunoprophylaxis for those at highest risk. While active vaccination strategies are finally available to protect adults 60 years and older from serious RSV infection, safe and effective vaccines for use in children have yet to emerge. In contrast, passive immunization strategies designed to protect all infants against RSV has finally met with success, with 2 new strategies approved by the US Food and Drug Administration during the second half of 2023. The first RSV passive immunization strategy to gain licensure for use in all infants is an extended half-life monoclonal antibody directed against an antigenic binding site on the RSV-F prefusion protein, a conformation not known to exist until 2013. The second novel passive immunization strategy approved during 2023 that has the potential to protect much of the infant population from RSV during young infancy centers on boosting preexisting RSV immunity during pregnancy using a prefusion RSV-F vaccine. The resulting boosted humoral immune response to RSV in the mother becomes part of the transplacental antibody endowment that is actively transported across the placenta to provide protection to those babies born at or near term. This review describes how and why these advances came to fruition seemingly "all at once" and provides insight into other passive immunization approaches that remain under development. [ABSTRACT FROM AUTHOR]

Alzheimer's disease, prevalent in individuals aged 60 and above, constitutes most dementia cases and significantly impairs memory and cognitive functions. With global Alzheimer's cases projected to triple by 2050, there is a pressing need for effective interventions. Lecanemab, a monoclonal antibody targeting amyloid-beta plaques, shows promise in slowing Alzheimer's progression. Positive clinical trial results have instilled hope in patients, prompting ongoing research to advance understanding and intervention possibilities. To contribute to this knowledge base, we conducted a systematic review and meta-analysis, focusing on lecanemab's efficacy and safety at a dosage of 10 mg/kg. This comprehensive approach aimed to address gaps in the current literature, scrutinize research disparities, and guide future investigations. Applying strict inclusion/exclusion criteria, we assessed study details, participant information, and intervention specifics, using the Cochrane risk of bias tool for quality evaluation. Statistical analyses, conducted with R software, included risk ratios and mean differences, assessing heterogeneity and publication bias. The meta-analysis reveals a significant positive effect of lecanemab (10 mg/kg biweekly) on cognitive outcomes in Alzheimer's disease. Consistent reductions in ADCOMS, CDR-SB, and ADAS-cog14 scores across studies indicate drug efficacy with narrow confidence intervals and no significant heterogeneity. While TEAE shows no significant difference, heightened risks of ARIA-E and ARIA-H associated with lecanemab underscore the need for vigilant safety monitoring in clinical practice. Despite the drug efficacy, the study emphasizes a balanced assessment of benefits and potential risks associated with lecanemab, providing critical insights for clinicians evaluating its use in addressing cognitive impairment in individuals with Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein's sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E. • Mouse monoclonal antibody recognizes 13-residue stretch in the HLA-E cytoplasmic tail. • mAb can be modified directly with fluorophores or biotin with sortase. • mAb stains HLA-E on tumor samples with high affinity and specificity. • mAb recognizes HLA-E in flow cytometry, immunoblot, IP, IHC, and IF. • mAb can be used as antibody-epitope tag for extracellular proteins. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors are the leading approaches in tumor immunotherapy. The aim of the study was to establish recommended phase 2 doses (RP2Ds) of intravenous cetrelimab, a checkpoint inhibitor, alone and with oral erdafitinib in Japanese patients with advanced solid tumors. This open-label, non-randomized, dose-escalation phase 1/1b study enrolled adults with advanced solid tumors who were ineligible for standard therapy. Study was conducted in two parts: phase 1a assessed cetrelimab at three dosing levels (80 mg every 2 weeks [Q2W], 240 mg Q2W, and 480 mg Q4W); phase 1b assessed cetrelimab+erdafitinib at two dosing levels (240 mg Q2W + 6 mg once daily [QD] and 240 mg Q2W + 8 mg QD). Primary endpoint was frequency and severity of dose-limiting toxicities (DLTs) of cetrelimab ± erdafitinib. In total 22 patients (phase 1a, n = 9; phase 1b, n = 13) were enrolled. Median duration of follow-up was 8.64 months in phase 1a and 2.33 months in phase 1b. In phase 1a, DLTs weren't reported while in phase 1b, 1 patient who received 240 mg cetrelimab + 6 mg erdafitinib reported Stevens-Johnson syndrome (grade 3, immune-related). Overall, 88.9% patients in phase 1a (grade ≥ 3: 44.4%) and 100.0% in phase 1b (grade ≥ 3: 53.8%) experienced ≥ 1 treatment-related adverse events (TEAEs); 33.3% in phase 1a and 38.5% in phase 1b reported serious TEAEs, of which 11.1% patients in phase 1a and 15.4% in phase 1b had TEAEs which led to treatment discontinuation. Cetrelimab alone and in combination with erdafitinib showed manageable safety in Japanese patients with advanced solid tumors. RP2Ds were determined as 480 mg cetrelimab Q4W for monotherapy, and cetrelimab 240 mg Q2W + erdafitinib 8 mg QD for combination therapy. [ABSTRACT FROM AUTHOR]

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

A continuous downstream process of monoclonal antibody was developed based on the process characterization. Periodic‐counter current chromatography (PCCC) with two protein A columns was used for the capture step. For low pH virus inactivation (VI), a batch reactor was employed, which can work as a surge (buffer) tank. Flow‐through chromatography (FTC) with two connected columns of different separation modes (anion‐mixed mode and cation exchange) was designed as a polish step. After 24 h PCCC run, the collected pool was processed for VI. After adjusting pH and electric conductivity, the solution was fed to the two connected FTC columns for 24 h. Virus filter was also connected to the exit of the connected‐column. PCCC and FTC were run in parallel. Six runs of different feed rates (0.5–10 L/day) and feed concentrations (1–3.2 g/L) were performed with protein A columns of 1–5 mL and FTC columns of 3–10 mL. The largest run (feed rate 10 L/day, feed concentration 2 g/L) was carried out at a GMP facility with 15 mL protein A columns and 100 mL FTC columns. Good recovery and purity values were obtained for all runs. The process was found to be flexible and stable for feed fluctuations. Only three surge or pool tanks were needed in addition to the final product pool tank. [ABSTRACT FROM AUTHOR]

The intensification of industrial biopharmaceutical production and the integration of process steps pave the way for patients to access affordable treatments. The predominantly batchwise biomanufacturing of established cell clarification technologies, stainless steel disc stack centrifugation (DSC) and single‐use (SU) depth filtration (DF), pose technological and economical bottlenecks, that include low biomass loading capacities and low product recoveries. Therefore, a novel SU‐based clarification platform was developed combining fluidized bed centrifugation (FBC) with integrated filtration. The feasibility of this approach was investigated for high cell concentration with more than 100E6 cells/mL. Furthermore, scalability to 200 L bioreactor scale was tested for moderate cell concentrations. In both trials, low harvest turbidities (4 NTU) and superior antibody recoveries (95%) were achieved. The impact on the overall economics of industrial SU biomanufacturing using an up‐scaled FBC approach was compared with DSC and DF technologies for different process parameters. As a result, the FBC showed to be the most cost‐effective alternative for annual mAb production below 500 kg. In addition, the FBC clarification of increasing cell concentrations was found to have minimal impact on overall process costs, in contrast to established technologies, demonstrating that the FBC approach is particularly suitable for intensified processes. [ABSTRACT FROM AUTHOR]

It has been reported that serum Clara cell secreted protein 16 (CC16) is a potential biomarker for lung injury diseases, but currently, there is no other method that is faster, more accurate, or more sensitive being applied in clinical practice apart from ELISA. The current study was designed to established a magnetic nanoparticles chemiluminescence immunoassay (MNPs-CLIA) for highly sensitive automated detection of serum Clara cell secretory protein 16 (CC16), and validated its diagnostic performance for lung disease.The study included the expression of CC16 recombinant protein, the preparation and screening of its monoclonal antibody (MAb), as well as the construction, optimization and analytical evaluation of the MNPs-CLIA method. The clinical application value of this method was investigated by detecting CC16 level in 296 serum samples.The linear range of the MNPs-CLIA assay system was 0.2–50 ng/mL, and the limit of detection was 0.037 ng/mL. Performance parameters such as specificity, recovery rate, and precision can meet the industry standards of in vitro diagnostic reagents. The established method reveals consistent results with ELISA (R2=0.9962) currently used clinically, and it also exhibits satisfactory diagnostic efficacy of silicosis, chronic obstructive pulmonary disease (COPD), and pulmonary sarcoidosis, with areas under the curve (AUC) of 0.9748, 0.8428 and 0.9128, respectively.Our established MNPs-CLIA method has the advantages of automation, high throughput, rapidity, and simplicity, and can be promoted for widely popularized in clinical applications. MNPs-CLIA detection of serum CC16 has efficient diagnostic potentiality for predicting and diagnosing lung diseases. [ABSTRACT FROM AUTHOR]

Background: The immunogenicity of the antigen-recognition domains of chimeric antigen receptor (CAR)-T cells leads to immune responses that may compromise the antitumor effects of the adoptively transferred T cells. Herein, we attempt to humanize a CD19-specific VHH (named H85) using in silico techniques and investigate the impact of antigen-recognition domain humanization on CAR expression and density, cytokine secretion, and cytolytic reactivity of CAR-T cells based on the humanized VHH. Methods: H85 was humanized (named HuH85), and then HuH85 was compared with H85 in terms of conformational structure, physicochemical properties, antigenicity and immunogenicity, solubility, flexibility, stability, and CD19-binding capacity using in silico techniques. Next, H85CAR-T cells and HuH85CAR-T cells were developed and CAR expression and surface density were assessed via flow cytometry. Ultimately, the antitumor reactivity and secreted levels of IFN-γ, IL-2, and TNF-α were assessed following the co-cultivation of the CAR-T cells with Ramos, Namalwa, and K562 cells. Results: In silico findings demonstrated no negative impacts on HuH85 as a result of humanization. Ultimately, H85CAR and HuH85CAR could be surface-expressed on transduced T cells at comparable levels as assessed via mean fluorescence intensity. Moreover, H85CAR-T cells and HuH85CAR-T cells mediated comparable antitumor effects against Ramos and Namalwa cells and secreted comparable levels of IFN-γ, IL-2, and TNF-α following co-cultivation. Conclusion: HuH85 can be used to develop immunotherapeutics against CD19-associated hematologic malignancies. Moreover, HuH85CAR-T cells must be further investigated in vitro and in preclinical xenograft models of CD19+ leukemias and lymphomas before advancing into clinical trials. [ABSTRACT FROM AUTHOR]

Asthma is a heterogeneous disease, and its development is the result of a combination of factors, including genetic factors, environmental factors, immune dysfunction and other factors. Its specific mechanism has not yet been fully investigated. With the improvement of disease models, research on the pathogenesis of asthma has made great progress. Immunological disorders play an important role in asthma. Previously, we thought that asthma was mainly caused by an imbalance between Th1 and Th2 immune responses, but this theory cannot fully explain the pathogenesis of asthma. Recent studies have shown that T-cell subsets such as Th1 cells, Th2 cells, Th17 cells, Tregs and their cytokines contribute to asthma through different mechanisms. For the purpose of the present study, asthma was classified into distinct phenotypes based on airway inflammatory cells, such as eosinophilic asthma, characterized by predominant eosinophil aggregates, and neutrophilic asthma, characterized by predominant neutrophil aggregates. This paper will examine the immune mechanisms underlying different types of asthma, and will utilize data from animal models and clinical studies targeting specific immune pathways to inform more precise treatments for this condition. [ABSTRACT FROM AUTHOR]

Background: Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP, or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk, thus potentially offering a new target for breast cancer treatment. Methods: We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration, invasion, and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice, Balb/c mice, and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity, we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. Results: Herein, we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone, named 12G2, which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth, was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions, 16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. Conclusions: Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases. [ABSTRACT FROM AUTHOR]

Since its initial report in 2015, CD47 has garnered significant attention as an innate immune checkpoint, raising expectations to become the next "PD-1." The optimistic early stages of clinical development spurred a flurry of licensing deals for CD47-targeted molecules and company mergers or acquisitions for related assets. However, a series of setbacks unfolded recently, starting with the July 2023 announcement of discontinuing the phase 3 ENHANCE study on Magrolimab plus Azacitidine for higher-risk myelodysplastic syndromes (MDS). Subsequently, in August 2023, the termination of the ASPEN-02 program, assessing Evorpacept in combination with Azacitidine in MDS patients, was disclosed due to insufficient improvement compared to Azacitidine alone. These setbacks have cast doubt on the feasibility of targeting CD47 in the industry. In this review, we delve into the challenges of developing CD47-SIRPα-targeted drugs, analyze factors contributing to the mentioned setbacks, discuss future perspectives, and explore potential solutions for enhancing CD47-SIRPα-targeted drug development. [ABSTRACT FROM AUTHOR]

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that causes substantial economic losses in the swine industry worldwide. The PDCoV NS6 protein is an accessory protein that plays a pivotal role in the viral life cycle and immune evasion. However, the functions of NS6 and its role in PDCoV pathogenesis remain largely unknown. In this study, we prepared a monoclonal antibody (mAb) 5-A11 that specifically recognizes the PDCoV NS6 protein. The mAb 5-A11 exhibited high specificity for PDCoV, with no cross-reactivity with several major porcine pathogenic viruses. Furthermore, the epitope recognized by mAb 5-A11 was precisely mapped to residues 70EYGSIYGKDFI80 of the NS6 protein using Western blot analysis. Notably, this epitope is highly conserved among different PDCoV isolates. Substantial variations were observed when comparing this epitope with the corresponding regions in the NS6 proteins of other δ coronaviruses, suggesting potential differences in the structure, function, and antigenicity of their NS6 proteins. Our findings provide valuable tools and insights for further elucidating the functions of the NS6 protein and its role in PDCoV pathogenesis, as well as for developing diagnostic and therapeutic strategies against PDCoV infection. [ABSTRACT FROM AUTHOR]

Background: The combination of immune checkpoint inhibitors with radiotherapy can enhance the immunomodulation by RT and reduce the growth of distant unirradiated tumors (abscopal effect); however, the results are still not very satisfactory. Therefore, new treatment options are needed to enhance this effect. Our previous study showed that the combination of Bifidobacterium (Bi) and its specific monoclonal antibody (mAb) could target and alleviate hypoxia at the tumor site and act as a radiosensitizer. In this study, we explored the anti-tumor efficacy of quadruple therapy (Bi + mAb and RT + αPD-1). The current study also aimed to probe into the complex immune mechanisms underlying this phenomenon. Methods: Constructed 4T1 breast and CT26 colon cancer tumor models. A comprehensive picture of the impact of constructed quadruple therapy was provided by tumor volume measurements, survival analysis, PET/CT imaging, immune cell infiltration analysis and cytokine expression levels. Results: The abscopal effect was further amplified in the "cold" tumor model and prolonged survival in tumor-bearing mice. Bi can colonized in primary and secondary tumors and direct the mAb to reach the tumor site, activate complement, enhance the ADCC effect and initiate the innate immune response. Then combined with αPD-1 and radiotherapy to stimulate adaptive immune response and synergize with cytokines to expand the immune efficacy and generate effective anti-tumor immune response. Conclusions: Bi was used as an artificially implanted anaerobic target to cause a transient "infection" at the tumor, causing the tumor to become locally inflamed and "hot", and at the same time, mAb was used to target Bi to enhance the local immune effect of the tumor, and then combined with radiotherapy and αPD-1 to amplify the abscopal effect in multiple dimensions. Therefore, the present study provided a new idea for the multipotent immune-activating function of antibody-targeted anaerobic bacteria for the RT treatment of extensively metastasized cancer patients. [ABSTRACT FROM AUTHOR]

The Strep Easy Kit, a bio‐enrichment dual ICG‐strip test, is a diagnostic tool designed for the detection of Streptococcus agalactiae, an important pathogenic bacterium in tilapia farming. The kit can simultaneously identify two different serotypes of S. agalactiae, serotype Ia and serotype III. This capability is crucial for the collection of valuable epidemiological data and facilitates strategic planning for effective vaccine development and deployment. The Strep Easy Kit consists of two main steps: pathogen enrichment and pathogen detection. The enrichment step increases the concentration of bacteria so that the bacterial load is raised to the level reliably detectable by the subsequent ICG strip test. This is achieved by incubating the fish samples in a suitable liquid medium under specified temperature and time conditions. The second step involves the use of the dual‐ICG strip test. This strip test consists of two monoclonal antibodies and one polyclonal antibody that are specific to S. agalactiae and can distinguish between S. agalactiae serotype Ia and S. agalactiae serotype III. This dual capability enables the ICG strip test to simultaneously detect both serotypes of S. agalactiae in a single test kit. The detection limit of the test kit, which consists of a dual ICG‐Strip test combined with an enrichment step, is 100 CFU/mL. The kit can be used to detect S. agalactiae in both live and dead fish samples, making it versatile for various testing scenarios. The test results obtained using the Strep Easy Kit have shown a 94.4% correlation with the standard method (Thai Agricultural Standard; TAS 10453‐2010), with 90.2% sensitivity and 100% specificity. Significant advantages of the Strep Easy Kit lie in its simplicity and portability, allowing farmers to perform the test by themselves and on‐site. This makes it a practical and accessible tool for the tilapia farming industry. [ABSTRACT FROM AUTHOR]

Background: The clinical benefit of coronavirus disease 2019 (COVID-19) treatments against new circulating variants remains unclear. We sought to describe characteristics and clinical outcomes of highest risk patients with COVID-19 receiving early COVID-19 treatments in Scotland. Methods: Retrospective cohort study of non-hospitalized patients diagnosed with COVID-19 from December 1, 2021–October 25, 2022, using Scottish administrative health data. We included adult patients who met ≥ 1 of the National Health Service highest risk criteria for early COVID-19 treatment and received outpatient treatment with sotrovimab, nirmatrelvir/ritonavir or molnupiravir, or no early COVID-19 treatment. Index date was defined as the earliest of COVID-19 diagnosis or early COVID-19 treatment. Baseline characteristics and acute clinical outcomes in the 28 days following index were reported. Values of ≤ 5 were suppressed. Results: In total, 2548 patients were included (492: sotrovimab, 276: nirmatrelvir/ritonavir, 71: molnupiravir, and 1709: eligible highest risk untreated). Patients aged ≥ 75 years accounted for 6.9% (n = 34/492), 21.0% (n = 58/276), 16.9% (n = 12/71) and 13.2% (n = 225/1709) of the cohorts, respectively. Advanced renal disease was reported in 6.7% (n = 33/492) of sotrovimab-treated and 4.7% (n = 81/1709) of untreated patients, and ≤ 5 nirmatrelvir/ritonavir-treated and molnupiravir-treated patients. All-cause hospitalizations were experienced by 5.3% (n = 25/476) of sotrovimab-treated patients, 6.9% (n = 12/175) of nirmatrelvir/ritonavir-treated patients, ≤ 5 (suppressed number) molnupiravir-treated patients and 13.3% (n = 216/1622) of untreated patients. There were no deaths in the treated cohorts; mortality was 4.3% (n = 70/1622) among untreated patients. Conclusions: Sotrovimab was often used by patients who were aged < 75 years. Among patients receiving early COVID-19 treatment, proportions of 28-day all-cause hospitalization and death were low. [ABSTRACT FROM AUTHOR]

Background: Orf, also known as contagious ecthyma (CE), is an acute, contagious zoonotic disease caused by the orf virus (ORFV). The F1L protein is a major immunodominant protein on the surface of ORFV and can induce the production of neutralizing antibodies. Methods: The prokaryotic expression system was used to produce the recombinant F1L protein of ORFV, which was subsequently purified and used to immunize mice. Positive hybridoma clones were screened using an indirect enzyme-linked immunosorbent assay (ELISA). The reactivity and specificity of the monoclonal antibody (mAb) were verified through Western blot and indirect immunofluorescence (IFA). The linear antigenic epitope specific to the mAb was identified through Western blot, using truncated F1L proteins expressed in eukaryotic cells. A multiple sequence alignment of the ORFV reference strains was performed to evaluate the degree of conservation of the identified epitope. Results: After three rounds of subcloning, a mAb named Ba-F1L was produced. Ba-F1L was found to react with both the exogenously expressed F1L protein and the native F1L protein from ORFV-infected cells, as confirmed by Western blot and IFA. The mAb recognized the core epitope 103CKSTCPKEM111, which is highly conserved among various ORFV strains, as shown by homologous sequence alignment. Conclusion: The mAb produced in the present study can be used as a diagnostic reagent for detecting ORFV and as a basic tool for exploring the mechanisms of orf pathogenesis. In addition, the identified linear epitope may be valuable for the development of epitope-based vaccines. [ABSTRACT FROM AUTHOR]

Soluble angiotensin‐converting enzyme 2 (ACE2) can act as a decoy molecule that neutralizes severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by blocking spike (S) proteins on virions from binding ACE2 on host cells. Based on structural insights of ACE2 and S proteins, we designed a "muco‐trapping" ACE2‐Fc conjugate, termed ACE2‐(G4S)6‐Fc, comprised of the extracellular segment of ACE2 (lacking the C‐terminal collectrin domain) that is linked to mucin‐binding IgG1‐Fc via an extended glycine‐serine flexible linker. ACE2‐(G4S)6‐Fc exhibits substantially greater binding affinity and neutralization potency than conventional full length ACE2‐Fc decoys or similar truncated ACE2‐Fc decoys without flexible linkers, possessing picomolar binding affinity and strong neutralization potency against pseudovirus and live virus. ACE2‐(G4S)6‐Fc effectively trapped fluorescent SARS‐CoV‐2 virus like particles in fresh human airway mucus and was stably nebulized using a commercial vibrating mesh nebulizer. Intranasal dosing of ACE2‐(G4S)6‐Fc in hamsters as late as 2 days postinfection provided a 10‐fold reduction in viral load in the nasal turbinate tissues by Day 4. These results strongly support further development of ACE2‐(G4S)6‐Fc as an inhaled immunotherapy for COVID‐19, as well as other emerging viruses that bind ACE2 for cellular entry. [ABSTRACT FROM AUTHOR]

Pulmonary drug delivery offers a minimally invasive and efficient method for treating lung conditions, leveraging the lungs' extensive surface area and blood flow for rapid drug absorption. Nebulized therapies aim to deliver drugs directly to the lung tissue. This study investigates the histological impact of nebulized tocilizumab—a monoclonal antibody targeting IL-6, traditionally administered intravenously for rheumatoid arthritis and severe COVID-19—on a murine model. Thirty BALB/c mice were nebulized with tocilizumab (10 mg, 5 mg, and 2.5 mg) and six controls were nebulized with saline solution. They were euthanized 48 h later, and their organs (lungs, nasal mucosa, and liver) were analyzed by a microscopic histological evaluation. The results indicate that all the mice survived the 48 h post-nebulization period without systemic compromise. The macroscopic examination showed no abnormalities, and the histopathological analysis revealed greater lung vascular changes in the control group than in the nebulized animals, which is attributable to the euthanasia with carbon dioxide. Additionally, increased alveolar macrophages were observed in the nebulized groups compared to controls. No significant histological changes were observed in the liver, indicating the safety of nebulized tocilizumab. In conclusion, these findings suggest the potential of nebulized tocilizumab for treating pulmonary inflammation, warranting further research to establish its efficacy and safety in clinical settings. [ABSTRACT FROM AUTHOR]

Simple Summary: We evaluated a novel proprietary murine B7-H3 antibody 8H8 for flow cytometric analyses of patients with acute myeloid leukemia (AML). The results showed substantial B7-H3 expression in a significant proportion of AML cases, particularly in the M5 group and in those with intermediate or poor risk profiles. The correlation between high B7-H3 expression and poorer overall and progression-free survival suggests that B7-H3 may serve as an additional promising negative prognostic marker to be considered in treatment decisions in AML. Despite recent advances in immunophenotyping, the prognosis of acute myeloid leukemia (AML) is still mainly estimated using age and genetic markers. As the genetic heterogeneity of AML patients is high, flow cytometry-based classification with appropriate biomarkers can efficiently complement risk stratification and treatment selection. An increased expression of B7-H3 (CD276), an immune checkpoint protein, has been reported and associated with poor prognosis. However, the available data are limited and heterogeneous. Here, we used a novel, proprietary murine anti-B7-H3 8H8 antibody for the flow cytometric analysis of B7-H3 expression in AML blasts from 77 patients. Our antibody reliably detected substantial B7-H3 expression in 62.3% of AML patients. B7-H3 expression was higher in the monocytic French–American–British (FAB) M5 group and in intermediate and poor risk patients according to the European Leukemia Network. Using receiver operating characteristics (ROCs), we identified a specific fluorescence intensity cut-off of 4.45 to discriminate between B7-H3high and B7-H3low expression. High B7-H3 expression was associated with shorter overall survival (OS) and progression-free survival (PFS). In conclusion, we have developed a novel B7-H3 antibody that serves as a new tool for the detection of B7-H3 expression in AML and may help to facilitate risk stratification and treatment selection in AML patients. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a main global epidemic increasing as population age and affecting approximately 10% of subjects over 45 years. COPD is a heterogeneous inflammatory disease with several endo-phenotypes and clinical presentations. Although neutrophilic inflammation is canonically considered a hallmark of COPD, eosinophilic inflammation can also be present in a subgroup of patients. Several other immune cells and cytokines play a key role in orchestrating and perpetuating the inflammatory pathways in COPD, making them attractive targets for treating this disorder. Recent studies have started to evaluate the possible role of type 2 (T2) inflammation and epithelial-derived alarmins (TSLP and IL-33) in COPD. Two phase III randomized clinical trials (RCTs) showed a modest reduction in exacerbations in COPD patients with eosinophilic phenotype treated with mepolizumab (anti-IL-5) or benralizumab (anti-IL-5Rα). A phase III RCT showed a 30% reduction in exacerbations in COPD patients with ≥ 300 eosinophils/μL treated with dupilumab (anti-IL-4Rα). These results suggest that blocking a single cytokine (e.g., IL-5) or its main target (i.e., IL-5Rα) is less promising than blocking a wider spectrum of cytokines (i.e., IL-4 and IL-13) in COPD. TSLP and IL-33 are upstream regulators of T2-high and T2-low immune responses in airway inflammation. Several ongoing RCTs are evaluating the efficacy and safety of anti-TSLP (tezepelumab), anti-IL-33 (itepekimab, tozorakimab), and anti-ST2 (astegolimab) in patients with COPD, who experience exacerbations. In conclusion, targeting T2 inflammation or epithelial-derived alarmins might represent a step forward in precision medicine for the treatment of a subset of COPD. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is considered the most common and prevalent form of dementia of adult-onset with characteristic progressive impairment in cognition and memory. The cure for AD has not been found yet and the treatments available until recently were only symptomatic. Regardless of multidisciplinary approaches and efforts made by pharmaceutical companies, it was only in the past two years that new drugs were approved for the treatment of the disease. Amyloid beta (A β) immunotherapy is at the core of this therapy, which is one of the most innovative approaches looking to change the course of AD. This technology is based on synthetic peptides or monoclonal antibodies (mAb) to reduce A β levels in the brain and slow down the advance of neurodegeneration. Hence, this article reviews the state of the art about AD neuropathogenesis, the traditional pharmacologic treatment, as well as the modern active and passive immunization describing approved drugs, and drug prototypes currently under investigation in different clinical trials. In addition, future perspectives on immunotherapeutic strategies for AD and the rise of the aptamer technology as a non-immunogenic alternative to curb the disease progression are discussed. Processing of APP to produce A β peptides which are the target of sequence-derived monoclonal antibodies currently approved for the treatment of AD. [Display omitted] [ABSTRACT FROM AUTHOR]

Precipitation during the viral inactivation, neutralization and depth filtration step of a monoclonal antibody (mAb) purification process can provide quantifiable and potentially significant impurity reduction. However, robust commercial implementation of this unit operation is limited due to the lack of a representative scale‐down model to characterize the removal of impurities. The objective of this work is to compare isoelectric impurity precipitation behavior for a monoclonal antibody product across scales, from benchtop to pilot manufacturing. Scaling parameters such as agitation and vessel geometry were investigated, with the precipitate amount and particle size distribution (PSD) characterized via turbidity and flow imaging microscopy. Qualitative analysis of the data shows that maintaining a consistent energy dissipation rate (EDR) could be used for approximate scaling of vessel geometry and agitator speeds in the absence of more detailed simulation. For a more rigorous approach, however, agitation was simulated via computational fluid dynamics (CFD) and these results were applied alongside a population balance model to simulate the trajectory of the size distribution of precipitate. CFD results were analyzed within a framework of a two‐compartment mixing model comprising regions of high‐ and low‐energy agitation, with material exchange between the two. Rate terms accounting for particle formation, growth and breakage within each region were defined, accounting for dependence on turbulence. This bifurcated model was successful in capturing the variability in particle sizes over time across scales. Such an approach enhances the mechanistic understanding of impurity precipitation and provides additional tools for model‐assisted prediction for process scaling. [ABSTRACT FROM AUTHOR]