Search Results (4,273)

No effective vaccines or treatments are currently available for severe fever with thrombocytopenia syndrome (SFTS), a fatal tick-borne infectious disease caused by the SFTS virus (SFTSV). This study evaluated the potential of ¹¹¹In-labeled anti-SFTSV antibodies targeting SFTSV structural proteins as single-photon emission computed tomography (SPECT) imaging agents for the selective visualization of SFTSV-infected sites. This study used nuclear medicine imaging to elucidate the pathology of SFTS and assess its therapeutic efficacy. Immunostaining experiments confirmed that the anti-SFTSV antibody (N-mAb), which targets the N protein, specifically accumulated in SFTSV-infected Vero E6 cells. ¹¹¹In-labeled N-mAb was successfully prepared using a diethylenetriaminepentaacetic acid (DTPA) chelator, resulting in [¹¹¹In]In-DTPA-N-mAb with high radiochemical purity exceeding 95% and a radiochemical yield of 55%. Cell-binding assays using SFTSV-infected Vero E6 cells demonstrated that [¹¹¹In]In-DTPA-N-mAb binding was detectable even without membrane permeabilization, with the binding intensity correlating with infection levels. In vivo studies using SFTSV-infected A129 mice showed high spleen accumulation of [¹¹¹In]In-DTPA-N-mAb (87.5% ID/g), consistent with SFTSV tropism, compared to 12.3% ID/g in mock-infected mice. SPECT/CT imaging clearly revealed high radioactivity in these regions. Although nonspecific accumulation was noted in the liver and spleen, this issue may be mitigated through antibody modifications such as fragmentation or PEGylation. Overall, [¹¹¹In]In-DTPA-N-mAb is a promising imaging agent for non-invasive visualization of SFTSV-infected sites and may aid in elucidating SFTS pathology and assessing therapeutic efficacy. Full article

Background/Objectives: Trastuzumab is an effective therapeutic intervention for treating HER2-positive breast cancers. The cost-effectiveness, global demand, and patent expiration of trastuzumab have led to the inflow of its biosimilars in the global market. With the rise of biosimilars in the biopharmaceutical market, it has become crucial to ensure that the biosimilar is at par with the original monoclonal antibody (mAb)in terms of efficacy, safety, and quality. Bioassay is one of the critical quality attributes (CQAs), hence developing a reliable and robust bioassay is essential for the evaluation of their biological activity and the harmonization of the quality of these biologics, supporting their safe and effective use in clinical practice. Methods: The present study aimed to develop a robust cell-based bioassay to assess the bioactivity of trastuzumab and its biosimilars for quality control testing. For this purpose, molecular characterization of different HER2-positive breast cancer cell lines of SKBR3, BT474, MDA-MD-453, MDA-MB-175, MCF-7, and MDA-MB-231 was performed to select a suitable cell line for the cell-based bioassay. Results: The SKBR3 cell line was found to express the HER2 receptors significantly higher in comparison to the other cell lines, and it was thereby selected for further bioassay optimization. The biological activity of trastuzumab was determined using the inhibition of proliferation (IOP) assay on the SKBR3, which was optimized based on the parameters of cell seeding density, drug dilution range, and incubation time, and it was further validated as per the compendial guidelines and found valid for the parameters of specificity, accuracy (% relative bias = 0.0067%), precision (repeatability: % GCV = 1.21%), linearity (R2 = 0.99), and range (50% to 200%). Additionally, the biological activity of different trastuzumab biosimilars was assessed using the validated IOP assay and compared to the HER2 binding assay performed by flow cytometry. The biological activity of different trastuzumab biosimilars was found to be comparable to the WHO primary reference standard of trastuzumab in terms of its relative potency using the IOP assay and binding assay by flow cytometry. Conclusions: Thus, an economic and robust cell-based bioassay method was successfully developed to assess the bioactivity of trastuzumab and its biosimilars. Full article

The ingestion of food contaminated with citrinin (CIT) poses a variety of health risks to humans and animals. The immunogens (CIT-COOH-BSA, CIT-H-BSA) and detection antigen (CIT-COOH-OVA, CIT-H-OVA) were synthesised using the active ester method (-COOH) and formaldehyde addition method (-H). A hybridoma cell line (3G5) that secretes anti-CIT monoclonal antibodies (mAbs) was screened via CIT-H-BSA immunisation of mice, cell fusion, and ELISA screening technology. The cell line was injected intraperitoneally to prepare ascites. The reaction conditions for the indirect competitive ELISA (ic-ELISA) were optimised, and an ic-ELISA method for detecting CIT was preliminarily established. The results revealed that the IC₅₀ of CIT from optimised ic-ELISA was 37 pg/mL, the linear detection range was 5.9~230 pg/mL, and the cross-reaction (CR) rate with other analogues was less than 0.01%. The intra-assay and interassay sample recovery rates of CIT were 84.7~92.0% and 83.6~91.6%, and the coefficients of variation (CVs) were less than 10%. The ic-ELISA of CIT established in this study was not significantly different from the HPLC results and is rapid, highly sensitive and strongly specific, providing technical support for the detection of CIT. Full article

The treatment of cancers with immunotherapies has yielded significant milestones in recent years. Amongst these immunotherapeutic strategies, the FDA has approved several checkpoint inhibitors (CPIs), primarily Anti-Programmed Death-1 (PD-1) and Programmed Death Ligand-1/2 (PDL-1/2) monoclonal antibodies, in the treatment of various cancers unresponsive to immune therapeutics. Such treatments resulted in significant clinical responses and the prolongation of survival in a subset of patients. However, not all patients responded to CPIs, due to various mechanisms of immune resistance. One such mechanism is that, in addition to PD-1 expression on CD8 T cells, other inhibitory receptors exist, such as Lymphocyte Activation Gene 3 (LAG-3), T cell Immunoglobulin Mucin 3 (TIM3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). These inhibitory receptors might be active in the presence of the above approved CPIs. Clearly, it is clinically challenging to block all such inhibitory receptors simultaneously using conventional antibodies. To circumvent this difficulty, we sought to target a potential transcription factor that may be involved in the molecular regulation of more than one inhibitory receptor. The transcription factor Yin Yang1 (YY1) was found to regulate the expression of PD-1, LAG-3, and TIM3. Therefore, we hypothesized that targeting YY1 in CD8 T cells should inhibit the expression of these receptors and, thus, prevent the inactivation of the anti-tumor CD8 T cells by these receptors, by corresponding ligands to tumor cells. This strategy should result in the prevention of immune evasion, leading to the inhibition of tumor growth. In addition, this strategy will be particularly effective in a subset of cancer patients who were unresponsive to approved CPIs. In this review, we discuss the regulation of LAG-3 by YY1 as proof of principle for the potential use of targeting YY1 as an alternative therapeutic approach to preventing the immune evasion of cancer. We present findings on the molecular regulations of both YY1 and LAG-3 expressions, the direct regulation of LAG-3 by YY1, the various approaches to targeting YY1 to evade immune evasion, and their clinical challenges. We also present bioinformatic analyses demonstrating the overexpression of LAG-3, YY1, and PD-L1 in various cancers, their associations with immune infiltrates, and the fact that when LAG-3 is hypermethylated in its promoter region it correlates with a better overall survival. Hence, targeting YY1 in CD8 T cells will result in restoring the anti-tumor immune response and tumor regression. Notably, in addition to the beneficial effects of targeting YY1 in CD8 T cells to inhibit the expression of inhibitory receptors, we also suggest targeting YY1 overexpressed in the tumor cells, which will also inhibit PD-L1 expression and other YY1-associated pro-tumorigenic activities. Full article

In spite of significant advancements in diagnosis and treatment, cancer remains one of the major threats to human health due to its ability to cause disease with high morbidity and mortality. A multifactorial and multitargeted approach is required towards intervention of the multitude of signaling pathways associated with carcinogenesis inclusive of angiogenesis and metastasis. In this context, plants provide an immense source of phytotherapeutics that show great promise as anticancer drugs. There is increasing epidemiological data indicating that diets rich in vegetables and fruits could decrease the risks of certain cancers. Several studies have proved that natural plant polyphenols, such as flavonoids, lignans, phenolic acids, alkaloids, phenylpropanoids, isoprenoids, terpenes, and stilbenes, could be used in anticancer prophylaxis and therapeutics by recruitment of mechanisms inclusive of antioxidant and anti-inflammatory activities and modulation of several molecular events associated with carcinogenesis. The current review discusses the anticancer activities of principal phytochemicals with focus on signaling circuits towards targeted cancer prophylaxis and therapy. Also addressed are plant-derived anti-cancer vaccines, nanoparticles, monoclonal antibodies, and immunotherapies. This review article brings to light the importance of plants and plant-based platforms as invaluable, low-cost sources of anti-cancer molecules of particular applicability in resource-poor developing countries. Full article

Background: The prediction of human clearance (CL) and subcutaneous (SC) bioavailability is a critical aspect of monoclonal antibody (mAb) selection for clinical development. While monkeys are a well-accepted model for predicting human CL, other preclinical species have been less-thoroughly explored. Unlike CL, predicting the bioavailability of SC administered mAbs in humans remains challenging as contributing factors are not well understood, and preclinical models have not been systematically evaluated. Methods: Non-clinical and clinical pharmacokinetic (PK) parameters were mined from public and internal sources for rats, cynomolgus monkeys, and humans. Intravenous (IV) and SC PK was determined in Sprague Dawley rats for fourteen mAbs without existing PK data. Together, we obtained cross-species data for 25 mAbs to evaluate CL and SC bioavailability relationships among rats, monkeys, and humans. Results: Rat and monkey CL significantly correlated with human CL and supported the use of species-specific exponents for body-weight-based allometric scaling. Notably, rat SC bioavailability significantly correlated with human SC bioavailability, while monkey SC bioavailability did not. Bioavailability also correlated with clearance. Conclusions: The rat model enables an early assessment of mAb PK properties, allowing discrimination among molecules in the discovery pipeline and prediction of human PK. Importantly, rat SC bioavailability significantly correlated with human SC bioavailability, which has not been observed with other species. Rats are cost-effective and efficient relative to monkeys and provide a valuable tool for pharmacokinetic predictions in therapeutic antibody discovery. Full article

The persistent threat of COVID-19, particularly with the emergence of new variants, underscores the urgency for innovative therapeutic strategies beyond conventional antiviral treatments. Current immunotherapies, including IL-6/IL-6R monoclonal antibodies and JAK inhibitors, exhibit suboptimal efficacy, necessitating alternative approaches. Our review delves into the significance of NAD+ metabolism in COVID-19 pathology, marked by decreased NAD+ levels and upregulated NAD+-consuming enzymes such as CD38 and poly (ADP-ribose) polymerases (PARPs). Recognizing NAD+’s pivotal role in energy metabolism and immune modulation, we propose modulating NAD+ homeostasis could bolster the host’s defensive capabilities against the virus. The article reviews the scientific rationale behind targeting NAD+ pathways for therapeutic benefit, utilizing strategies such as NAD+ precursor supplementation and enzyme inhibition to modulate immune function. While preliminary data are encouraging, the challenge lies in optimizing these interventions for clinical use. Future research should aim to unravel the intricate roles of key metabolites and enzymes in NAD+ metabolism and to elucidate their specific mechanisms of action. This will be essential for developing targeted NAD+ therapies, potentially transforming the management of COVID-19 and setting a precedent for addressing other infectious diseases. Full article

Background/Objectives: SARS-CoV-2 was responsible for the global pandemic. Approximately 10–15% of patients with COVID-19 developed respiratory failure with adult acute respiratory distress syndrome (ARDS), which required treatment in the Intensive Care Unit (ICU). The cytokine storm observed in severe COVID-19 was frequently handled with steroids. Synergically, tocilizumab, an anti-interleukin-6 receptor monoclonal antibody, gained popularity as a cytokine storm-suppressing agent. However, immunosuppression was proven to increase the predisposition to infections with resistant bacteria. Our study aimed to assess the relationship between positive tests for secondary infections and the survival of patients with severe COVID-19-attributed ARDS treated with immunosuppressive agents. Methods: This study included 342 patients qualified for the ICU and mechanical ventilation (MV). The patients were divided based on the type of immunomodulating therapy and the culture tests results. Results: The results showed the highest survival rate among patients <61 years, favoring the combined treatment (tocilizumab + steroids). Atrial fibrillation (AF) and coronary heart disease (CHD) correlated with a lower survival rate than other comorbidities. Tocilizumab was associated with an increased risk of positive pathogen cultures, which could potentially cause secondary infections; however, the survival rate among these patients was higher. Conclusions: MV and ICU procedures as well as the application of tocilizumab significantly decreased the mortality rate in patients with severe COVID-19-related ARDS. The suppression of cytokine storms played a crucial role in survival. Tocilizumab was found to be both efficient and safe despite the ‘side effect’ of the increased risk of positive results for secondary infections. Full article

Background/Objectives: Vedolizumab (VDZ) is approved in the treatment of patients with moderate to severe ulcerative colitis (UC) or Crohn’s disease (CD). VDZ exhibits considerable variability in its pharmacokinetic (PK) profile, and its exposure-response relationship is not yet fully understood. The aim was to investigate the variability in VDZ trough levels and PK parameters, to assess the relationship between VDZ PK and biochemical response, as well as clinical and endoscopic outcomes. Methods: We included 61 UC and 45 CD patients. Patients’ data and trough VDZ concentrations were retrospectively obtained. Population PK analysis was performed using non-linear mixed-effects modelling with NONMEM (version 7.5). Graphs and statistical analyses were performed using R (version 4.1.3). Results: In total, 116 trough VDZ concentrations from 106 patients were described by a two-compartment model. For a typical patient, clearance (CL) was estimated at 0.159 L/day, while in patients previously treated with anti-TNFα agents, VDZ CL increased by 26.4% on average. In univariate binary logistic regression, VDZ trough concentration was not statistically significant predictor of remission, whereas CL was. Moreover, combined CL and faecal calprotectin (FCP) were a statistically significant predictors of remission. The hazard ratio (HR) for CL above 0.1886 L/day was 0.35 (p = 0.05) and for FCP below 250 µg/g was 2.66 (p = 0.02) in a time-to-event analysis. Conclusions: Our population PK model incorporates the effect of prior anti-TNFα agents on CL, suggesting its association with more severe forms of IBD. VDZ CL emerged as a more robust and clinically relevant predictor of remission in IBD patients than trough concentration. Full article

Natural killer (NK) and CD8⁺ T cell function is compromised in human immunodeficiency virus type 1 (HIV-1) infection by increased expression of inhibitory receptors such as TIGIT (T cell immunoreceptor with Ig and ITIM domains). Blocking inhibitory receptors or their ligands with monoclonal antibodies (mAb) has potential to improve antiviral immunity in general and facilitate HIV eradication strategies. We assessed the impact of TIGIT engagement and blockade on cytotoxicity, degranulation, and interferon-gamma (IFN-γ) production by CD8⁺ T cells from persons living with HIV (PLWH). The effect of TIGIT engagement on non-specific anti-CD3-redirected cytotoxicity was assessed in redirected cytotoxicity assays, and the effect of TIGIT blockade on HIV-specific CD8⁺ T cell responses was assessed by flow cytometry. In 14/19 cases where peripheral blood mononuclear cells (PBMC) mediated >10% redirected cytotoxicity, TIGIT engagement reduced the level of cytotoxicity to <90% of control values. We selected PLWH with >1000 HIV Gag or Nef-specific IFN-γ spot forming cells per million PBMC to quantify the effects of TIGIT blockade on HIV-specific CD8⁺ T cell responses by flow cytometry. Cell surface TIGIT expression decreased on CD8⁺ T cells from 23/40 PLWH following TIGIT blockade and this loss was associated with increased anti-TIGIT mAb fluorescence on monocytes. In total, 6 of these 23 PLWH had enhanced HIV-specific CD8⁺ T cell degranulation and IFN-γ production with TIGIT blockade, compared to 0/17 with no decrease in cell surface TIGIT expression. Reduced CD8⁺ T cell TIGIT expression with TIGIT blockade involved trogocytosis by circulating monocytes, suggesting that an effector monocyte population and intact fragment crystallizable (Fc) functions are required for mAb-based TIGIT blockade to effectively enhance HIV-specific CD8⁺ T cell responses. Full article

Epidermal growth factor receptor (EGFR) inhibition is crucial in treating RAS wild-type metastatic colorectal cancer, yet current testing methods may miss rare RAS variants affecting treatment efficacy. We analyzed 4122 colorectal cancer patients receiving anti-EGFR antibodies from the Center for Cancer Genomics and Advanced Therapeutics database, identifying 54 patients (1.3%) with rare RAS variants undetectable by standard testing. These patients showed significantly lower response rates to anti-EGFR therapy (28.3%) compared to RAS wild-type cases (44.6%, p = 0.003). Disease control rates were also lower in rare variant cases (60.9%) versus wild-type cases (80.0%). Most common rare variants included KRAS Q22K, A59E, and A11_G12insGA. Comprehensive genomic profiling revealed additional alterations in TP53 (90.7%), APC (87.0%), and non-V600E BRAF mutations (25.9%). Our findings suggest that rare RAS variants predict poor anti-EGFR therapy response, highlighting the potential benefit of comprehensive genomic profiling before treatment initiation. This study provides real-world evidence supporting the clinical relevance of rare RAS variants in treatment decision-making for colorectal cancer. Future studies should focus on developing cost-effective comprehensive testing strategies and evaluating alternative treatment approaches for patients with rare RAS variants. Full article

Antibodies are complex protein structures, and producing them using eukaryotic expression systems presents significant challenges. One frequently overlooked aspect of expression vectors is the nucleotide sequence encoding the signal peptide, which plays a pivotal role in facilitating the secretion of recombinant proteins. This study presents the development of an integrative vector, pVEAL3, for expressing full-length recombinant monoclonal antibodies in mammalian cells. The vector features a distinctive nucleotide sequence that encodes an artificial chimeric signal peptide with the following amino acid sequence: MMRTLILAVLLVYFCATVHC. Additionally, the vector incorporates several regulatory elements to enhance antibody expression, including the Gaussia luciferase signal sequence, internal ribosome entry site (IRES), P2A peptide, and a furin cleavage site. These elements coordinate to regulate the synthesis levels of the antibody chains. The analysis of clones obtained via transfection with the developed vector showed that over 95% of them secreted antibodies at levels significantly higher than those of the control. The immunochemical analysis of the chimeric antibody produced by the CHO-K1-10H10ch cell line confirmed the preservation of its functional activity. Full article

Background: Monoclonal antibodies approved by the FDA, lecanemab, donanemab, and aducanumab, are failing to meet the expected efficacy to treat early Alzheimer’s disease, and aducanumab has been recalled. Methods: Recently, it was reported that the clinical trials of these antibodies may have violated patient’s rights and subjected them to high, likely lethal risk. The challenge with developing antibodies to treat neurological disorders is their poor blood–brain barrier (BBB) penetration if the antibody must enter the brain, resulting in almost negligible brain bioavailability, requiring high dosing that can be toxic. Results: The reported efficacy of these drugs should also be reviewed, considering the placebo effects, since all antibodies have shown severe side effects that are not prevented by the placebo responses. In this critical and urgent advice to the FDA, I am suggesting a guideline amendment to all clinical trials requiring proof of sufficient brain bioavailability at the site of action, where it is known. Conclusions: For antibodies to cross the blood–brain barrier, there are proven options such as conjugating with transferrin protein, making clinical trials in its absence more questionable. Full article

Der p1 is one of the major allergens causing house dust mite (HDM) allergy. Pathological Der p1-specific B cells play a key role in allergic inflammation as producers of allergen-specific antibodies. Crosslinking the inhibitory FcγRIIb with the B cell receptor triggers a high-affinity suppressive signal in B cells. Selective elimination of allergen-specific cells could potentially be achieved by administering chimeric molecules that combine, through protein engineering, the FcγRIIb-targeting monoclonal 2.4G2 antibody with the epitope-carrying Dp52–71 peptides from Der p1. We tested this hypothesis, in a chronic mouse model of HDM allergy induced in BalB/c mice, using FACS and ELISA assays, along with histopathological and correlational analyses. Dp52–71chimera treatment of HDM-challenged mice led to a decrease in serum anti-HDM IgG1 antibodies, a reduction in BALF β-hexosaminidase levels, a lowered number of SiglecF^high CD11c^low eosinophils, and an improved lung PAS score. Furthermore, we observed overexpression of FcγRIIb on the surface of CD19 cells in the lungs of HDM-challenged animals, which negatively correlated with the levels of lung alveolar macrophages, neutrophils, and BALF IL-13. Taken together, these results suggest that the use of FcγRIIb overexpression, combined with the expansion of chimeric protein technology to include more epitopes, could improve the outcome of inflammation. Full article

The livestock industry uses ofloxacin, an antibiotic, to prevent several animal diseases; however, the overdose of ofloxacin used in animal farming treatments may appear in food products and cause some adverse human health effects. Hence, there is an immediate need to develop a method suitable for on site large-scale detection of ofloxacin residues in animal-derived foods. This study aimed to prepare a monoclonal antibody with high sensitivity and affinity for ofloxacin by re-synthesizing the ofloxacin hapten and synthesizing the corresponding complete antigen. The IC₅₀ of the enzyme-linked immunosorbent assay (ic-ELISA) was 0.13 ng/mL, and the detection limit was 0.033 ng/mL. The visual detection limit of the established colloidal gold immunochromatographic test strip, for the visual detection of actual samples, was 1 ng/g. In summary, this work establishes a rapid detection method of ofloxacin residues on the basis of colloidal gold immunochromatography that is suitable for actual detection. Full article