Search Results (236)

Background/Objectives: High-risk neuroblastoma patients are treated with approved anti-ganglioside GD2 antibodies of moderate (dinutuximab beta; DB) and higher binding affinity (naxitamab; NAXI). We evaluated the functional potency of DB compared to NAXI and investigated the target-mediated drug disposition (TMDD). Methods: Tumor spheroids were generated from neuroblastoma cells with varying GD2 expression, stably expressing iRFP680 as a viability marker. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) were assessed in a long-term life-cell viability assay using serial dilutions of the GD2 antibodies. Binding activity was determined by flow cytometry. Processes involved in TMDD were analyzed, including antibody binding to dead tumor cells and to soluble GD2 (sGD2), antibody internalization into tumor and immune cells and the impact of sGD2 on DB and NAXI-mediated ADCC. Results: DB and NAXI mediated a concentration-dependent ADCC response against GD2-positive spheroids and no response against GD2-negative spheroids. DB showed a significantly higher ADCC potency than NAXI in all GD2-positive spheroid models. Binding activity of DB and NAXI was not significantly different. However, the decrease of anti-GD2 antibody binding to viable GD2-positive tumor cells following co-incubation with dead GD2-positive tumor cells or sGD2 was significantly higher for NAXI than DB. Additionally, we found an increased internalization of NAXI compared to DB by tumor cells and particularly CD64+ monocytes. Finally, sGD2 impaired NAXI-mediated ADCC to a significantly greater extent than DB-mediated ADCC. Conclusions: DB has a higher ADCC potency over NAXI at clinically relevant concentrations, attributed to stronger TMDD effects of NAXI compared to DB. Full article

Objectives: Understanding the antibody response in monkeypox virus (MPXV)-infected and uninfected individuals is essential for developing next-generation MPXV vaccines. This study aimed to characterize neutralizing antibody (NAb) and antibody-dependent cellular cytotoxicity (ADCC) responses in both groups, providing insights into immune protection and vaccine design. Methods: A recombinant vaccinia Tian Tan (VTT) virus was utilized to develop high-throughput luciferase-reporter-based neutralization and ADCC assays. These assays were applied to evaluate the presence and levels of poxvirus-specific antibodies in MPXV-infected and uninfected individuals, including those vaccinated with vaccinia-based vaccines. Results: Poxvirus-specific NAbs were detected in MPXV-negative individuals with prior vaccinia vaccination. However, MSM individuals exhibited significantly lower pre-existing NAb levels than non-MSM individuals, potentially contributing to their higher susceptibility to MPXV infection. In individuals with mild MPXV infection, robust NAb and ADCC responses were observed, regardless of vaccination status. Additionally, HIV-positive individuals demonstrated comparable antibody responses following MPXV infection. Conclusions: These findings highlight the potential role of pre-existing NAbs in MPXV susceptibility and the strong immune response elicited by mild MPXV infection. Further research is needed to determine whether MPXV-specific antibodies mitigate disease progression, which could inform the development of effective MPXV vaccines. Full article

Virus-like nanoparticles (VNPs) based on Moloney murine leukemia virus represent a well-established platform for the expression of heterologous molecules such as cytokines, cytokine receptors, peptide MHC (pMHC) and major allergens, but their application for inducing protective anti-viral immunity has remained understudied as of yet. Here, we variably fused the wildtype SARS-CoV-2 spike, its receptor-binding domain (RBD) and nucleocapsid (NC) to the minimal CD16b-GPI anchor acceptor sequence for expression on the surface of VNP. Moreover, a CD16b-GPI-anchored single-chain version of IL-12 was tested for its adjuvanticity. VNPs expressing RBD::CD16b-GPI alone or in combination with IL-12::CD16b-GPI were used to immunize BALB/c mice intramuscularly and subsequently to investigate virus-specific humoral and cellular immune responses. CD16b-GPI-anchored viral molecules and IL-12-GPI were well-expressed on HEK-293T-producer cells and purified VNPs. After the immunization of mice with VNPs, RBD-specific antibodies were only induced with RBD-expressing VNPs, but not with empty control VNPs or VNPs solely expressing IL-12. Mice immunized with RBD VNPs produced RBD-specific IgM, IgG_2a and IgG₁ after the first immunization, whereas RBD-specific IgA only appeared after a booster immunization. Protein/peptide microarray and ELISA analyses confirmed exclusive IgG reactivity with folded but not unfolded RBD and showed no specific IgG reactivity with linear RBD peptides. Notably, booster injections gradually increased long-term IgG antibody avidity as measured by ELISA. Interestingly, the final immunization with RBD–Omicron VNPs mainly enhanced preexisting RBD Wuhan Hu-1-specific antibodies. Furthermore, the induced antibodies significantly neutralized SARS-CoV-2 and specifically enhanced cellular cytotoxicity (ADCC) against RBD protein-expressing target cells. In summary, VNPs expressing viral proteins, even in the absence of adjuvants, efficiently induce functional SARS-CoV-2-specific antibodies of all three major classes, making this technology very interesting for future vaccine development and boosting strategies with low reactogenicity. Full article

Deep learning models such as convolutional neural networks (CNNs) and vision transformers (ViTs) perform well in histological image classification, but often lack interpretability. We introduce a unified framework that adds an attention branch and CAM Fostering, an entropy-based regularizer, to improve Grad-CAM visualizations. Six backbone architectures (ResNet-50, DenseNet-201, EfficientNet-b0, ResNeXt-50, ConvNeXt, CoatNet-small) were trained, with and without our modifications, on five H&E-stained datasets. We measured explanation quality using coherence, complexity, confidence drop, and their harmonic mean (ADCC). Our method increased the ADCC in five of the six backbones; ResNet-50 saw the largest gain (+15.65%), and CoatNet-small achieved the highest overall score (+2.69%), peaking at 77.90% on the non-Hodgkin lymphoma set. The classification accuracy remained stable or improved in four models. These results show that combining attention and entropy produces clearer, more informative heatmaps without degrading performance. Our contributions include a modular architecture for both convolutional and hybrid models and a comprehensive, quantitative explainability evaluation suite. Full article

Background/Objectives: Chimeric antigen receptor T-cell (CAR-T) therapy is a novel form of adoptive cellular immunotherapy that involves modifying autologous T cells to recognize and target tumor-associated antigens (TAAs) on malignant cells, independent of major histocompatibility complex (MHC) restriction. Although CAR-T therapy has shown remarkable success in treating hematologic malignancies, its efficacy in solid tumors remains limited, largely due to the lack of tumor-specific antigens and the complexity of the tumor microenvironment. This review aims to explore the rationale for continuing the development of adoptive cellular therapies in head and neck cancer (HNC), offering insights into the diagnostic and therapeutic challenges associated with this heterogeneous group of malignancies. Methods: We conducted a comprehensive literature review using the PubMed database to identify relevant studies on the application of CAR-T cell therapy in the management of HNC. Results: HNC presented numerous barriers to CAR-T cell infiltration, primarily due to the unique characteristics of its tumor microenvironment (TME). The TME in HNC is notably immunosuppressive, with a lymphocytic infiltrate predominantly composed of regulatory T cells (Tregs) and natural killer (NK) cells. These immune cells typically exhibit low expression of the CD16 receptor, which plays a crucial role in mediating antibody-dependent cellular cytotoxicity (ADCC), thereby limiting the effectiveness of CAR-T cell therapy. Conclusions: This comprehensive review suggests a potential clinical applicability of CAR-T therapy in HNC management. Full article

Background: Administration of PARP inhibitors against breast and ovarian cancers with BRCA1 and BRCA2 mutations has shown clinical benefits in patients. However, these agents are also toxic and have a narrow therapeutic index. Objectives: In this work, we aimed to identify membrane proteins that are specifically upregulated in these cancers. Methods: We interrogated public datasets to analyze genes upregulated or downregulated when these mutations were present, compared with wild-type cancers. Surface protein expression and functional annotation analyses were also performed. Results: In breast cancer, we identified 11 upregulated and 44 downregulated transcripts in BRCA1-mut, while 10 upregulated and 57 downregulated transcripts were identified in BRCA2-mut cancers. In ovarian cancer, 79 transcripts were upregulated and 123 were downregulated in BRCA1-mut cancers, while five were upregulated and seven were downregulated in BRCA2-mut tumors. Regarding the biological function related to these genes, in BRCA1-mutated ovarian cancers, the main functions of upregulated genes included MHC assembly or regulation of the interferon gamma pathway; in BRCA2-mut ovarian cancers, regulation of phosphorylation and signaling; in BRCA1-mut breast cancers, cell damage repair and angiogenesis; and finally, in BRCA2-mut breast cancers, cytokine production and T-cell migration. Genes expressed in the surface membrane or extracellular matrix and related to patient outcomes included B3GNT7 and CTSV in BRCA2-mut breast cancers, exhibiting detrimental prognoses. CD6, CXCL9, and CXCL13 were associated with favorable outcomes in BRCA1-mutant ovarian cancers. The last three genes were also correlated with the infiltration of effector T cells and dendritic cells in ovarian tumors. Conclusions: In summary, we identified deregulated candidate genes that could be used as therapeutic targets. Full article

The resistance of breast cancer cells to therapeutic antibodies such as anti-HER2 trastuzumab can be overcome by engaging natural killer (NK) cells for killing antibody-binding tumor cells via antibody-dependent cellular cytotoxicity (ADCC). Here, we investigated how autophagy modulation affects trastuzumab-mediated ADCC in HER2-positive JIMT1 breast cancer cells and NK cells. Autophagy inducers (rapamycin and resveratrol) had no significant impact, but the inhibitor bafilomycin nearly abolished ADCC. Protection occurred when either cancer or NK cells were pretreated, indicating dual effects. Bafilomycin reduced phosphatidylserine externalization, the loss of plasma membrane integrity, caspase-3/7 activity, and DNA fragmentation. It downregulated pro-apoptotic BAK1 and BAX without altering BCL-2. Additionally, bafilomycin decreased HER2 surface expression, impairing trastuzumab binding, and modulated immune regulators (STAT1, CD95, and PD-L1) in NK and/or in the cancer cells. Bafilomycin disrupted HER2 trafficking and induced HER2 internalization, leading to its accumulation in cytoplasmic vesicles. These findings show that autophagy inhibition by bafilomycin confers ADCC resistance by altering apoptosis, immune signaling, and HER2 dynamics. The study underscores autophagy’s role in antibody-based cancer therapy efficacy. Full article

Background: Recombinant monoclonal antibodies represent a vital category of biologics, constituting the largest class of molecules used to treat autoimmune disorders, cancers, rheumatoid arthritis, and other chronic conditions. The IgG1 subclass is the most potent among all the immunoglobulin gamma (IgG) antibodies, inducing Fc-related effector functions. N-linked glycan distribution of therapeutic IgG1s affects Fc-related effector functions such as CDC (complement-dependent cytotoxicity) and ADCC (antibody dependent cell-mediated cytotoxicity) biological activities and efficacy in vivo. Hence, as a critical quality attribute (CQA), the glycosylation profile of therapeutic IgG1s must be consistently preserved, which is primarily influenced by manufacturing process factors. In the era of biosimilars, it is challenging for biopharmaceutical manufacturers to not only obtain the desired glycan distribution consistently but also to meet the innovator molecule specifications as per the regulatory agencies. Methods: This study investigates the CHO fed-batch process parameters that affect the titer and terminal galactosylation of the TNF-α blocker-IgG1. It was hypothesized that galactose supplementation would enhance the galactosylation of TNF-α blocker-IgG1. Results: It was observed that such in-cultivation process shift does not affect cell culture parameters yet significantly enhances the galactosylation of TNF-α blocker-IgG1. Interestingly, the results indicate that supplementing D-galactose from the exponential phase of the CHO fed-batch process had the greatest effect on Fc galactosylation, increasing the amount of total galactosylated TNF-α blocker-IgG1 from 7.7% to 15.8%. Conclusions: Our results demonstrate a relatively easy and viable technique for cell culture engineering that is more appropriate for industrial production than costly in vitro glycoengineering. Full article

Mogamulizumab is a humanized monoclonal antibody that targets C-C chemokine receptor 4 (CCR4) present on certain T cells in lymphomas and leukemias. This antibody-based therapy has demonstrated efficacy in treating various cutaneous T cell lymphomas (CTCLs), including mycosis fungoides and Sézary syndrome, through the depletion of CCR4-expressing T cells by antibody-dependent cellular cytotoxicity (ADCC). However, the precise epitope and binding mode of mogamulizumab responsible for its augmented ADCC activity remain undisclosed. Here, X-ray crystallographic studies of mogamulizumab in complex with a 28-residue N-terminal peptide indicated that SIYSNYYLYES (residues 14–24) would constitute the antibody epitope. Another high-resolution structure, using a short core peptide of these 11 residues, has elucidated unambiguous electron density for the bound peptide, confirming consistent binding for both peptides. This linear epitope is located in the membrane-proximal region of CCR4, facilitating the Fc-mediated effector functions, including ADCC. The structures also provide insights into the molecular basis for the resistance of the CCR4 L21V variant to mogamulizumab, which is due to a lack of structural complementarity with mogamulizumab binding. Understanding the structural basis for the mechanism of action of mogamulizumab is crucial for optimizing anti-CCR4 therapeutics to improve treatment outcomes for patients with these challenging diseases. Full article

Cases of new COVID-19 infection, which manifested in 2019 and caused a global socioeconomic crisis, still continue to be registered worldwide. The high mutational activity of SARS-CoV-2 leads to the emergence of new antigenic variants of the virus, which significantly reduces the effectiveness of COVID-19 vaccines, as well as the sensitivity of diagnostic test systems based on variable viral antigens. These problems may be solved by focusing on highly conserved coronavirus antigens, for example nucleocapsid (N) protein, which is actively expressed by coronavirus-infected cells and serves as a target for the production of virus-specific antibodies and T cell responses. It is known that anti-N antibodies are non-neutralizing, but their protective potential and functional activity are not sufficiently studied. Here, the protective effect of anti-N antibodies was studied in Syrian hamsters passively immunized with polyclonal sera raised to N(B.1) recombinant protein. The animals were infected with 10⁵ or 10⁴ TCID₅₀ of SARS-CoV-2 (B.1, Wuhan or BA.2.86.1.1.18, Omicron) 6 h after serum passive transfer, and protection was assessed by weight loss, clinical manifestation of disease, viral titers in the respiratory tract, as well as by the histopathological evaluation of lung tissues. The functional activity of anti-N(B.1) antibodies was evaluated by complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) assays. The protection of anti-N antibodies was evident only against a lower dose of SARS-CoV-2 (B.1) challenge, whereas almost no protection was revealed against BA.2.86.1.1.18 variant. Anti-N(B.1) monoclonal antibodies were able to stimulate both CDC and ADCC. Thus, anti-N(B.1) antibodies possess protective activity against homologous challenge infection, which is possibly mediated by innate Fc-mediated immune reactions. These data may be informative for the development of N-based broadly protective COVID-19 vaccines. Full article

Background: This paper will identify the potential genetic causes of multimorbidity associated with autosomal dominant congenital cataract (ADCC). Methods: Whole exome sequencing (WES) was performed on 13 individuals affected with ADCC. Subsequent bioinformatic analyses identified variants with deleterious pathogenicity scores. Results: Disease-causing variants were identified in 8 genes already linked to cataract (CHMP4B, CRYAA, CRYBA1, CRYGD, CYP21A2, GJA8, OPA1, and POMGNT1), but variants previously associated with systemic disorders were also found in a further 11 genes (ACTL9, ALDH18A1, CBS, COL4A3, GALT, LRP5, NOD2, PCK2, POMT2, RSPH4A, and SMO). All variants were identified via pipeline data analysis, prioritising rare coding variants using Kaviar and the Genome Aggregation Database. The following ADCC-associated non-ocular phenotypes were identified in four patients in the cohort: (i) Horner’s pupils, vaso-vagal syncope, and paroxysmal orthostatic tachycardia syndrome; (ii) reduced kidney function and high cholesterol; (iii) hypertension, high cholesterol, and kidney stones; and (iv) grade 1 spondylolysis. Conclusions: We report 11 novel genes identified in an ADCC patient cohort associated with systemic disorders found, along with 8 known cataract-causing genes. Our findings broaden the spectrum of potentially cataract-associated genes and their related lens phenotypes, as well as evidence multimorbidities in four patients, highlighting the importance of careful multisystem phenotyping following genetic analysis. Full article

Disease progression and treatment resistance in colorectal and other cancers are driven by a subset of cells within the tumor that have stem-cell-like properties and long-term tumorigenic potential. These stem-cell-like cells express the leucine-rich G repeat-containing protein-coupled receptor 5 (LGR5) and have characteristics similar to tissue-resident stem cells in normal adult tissues such as the colon. Organoid models of murine and human colorectal and other cancers contain LGR5-expressing (LGR5+) stem-cell-like cells and can be used to investigate the underlying mechanisms of cancer development, progression, therapy vulnerability, and resistance. A large biobank of organoids derived from colorectal cancer or adjacent normal tissue was developed. We performed a large-scale unbiased functional screen to identify bispecific antibodies (BsAbs) that preferentially inhibit the growth of colon tumor-derived, as compared to normal tissue-derived, organoids. We identified the most potent BsAb in the screen as petosemtamab, a Biclonics^® BsAb targeting both LGR5 and the epidermal growth factor receptor (EGFR). Petosemtamab employs three distinct mechanisms of action: EGFR ligand blocking, EGFR receptor internalization and degradation in LGR5+ cells, and Fc-mediated activation of the innate immune system by antibody-dependent cellular phagocytosis (ADCP) and enhanced antibody-dependent cellular cytotoxicity (ADCC) (see graphical abstract). Petosemtamab has demonstrated substantial clinical activity in recurrent/metastatic head and neck squamous cell carcinoma (r/m HNSCC). The safety profile is generally favorable, with low rates of skin and gastrointestinal toxicity. Phase 3 trials are ongoing in both first-line programmed death-ligand 1-positive (PD-L1+) and second/third-line r/m HNSCC. Full article

Monoclonal antibodies (mAbs) have become a cornerstone in the treatment of follicular lymphoma (FL), offering highly specific therapeutic targeting that enhances efficacy while minimizing systemic toxicity. Their mechanisms of action include antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and direct apoptotic signaling, effectively mediating malignant B-cell depletion. Anti-CD20 mAbs, such as rituximab and obinutuzumab, have significantly improved progression-free survival (PFS) and overall survival (OS), establishing immunochemotherapy as the standard of care for FL. However, the emergence of treatment resistance, often characterized by CD20 antigen downregulation or immune escape, has prompted the development of next-generation mAbs with enhanced effector functions. Bispecific antibodies (BsAbs), which simultaneously engage CD20-expressing tumor cells and CD3-positive cytotoxic T cells, have emerged as a novel immunotherapeutic strategy, redirecting T-cell activity to eliminate malignant B cells independently of major histocompatibility complex (MHC) antigen presentation. Additionally, antibody–drug conjugates (ADCs) offer a targeted cytotoxic approach by delivering potent chemotherapeutic payloads directly to tumor cells while limiting off-target effects. The integration of mAbs with immune checkpoint inhibitors and immunomodulatory agents is further enhancing treatment outcomes by overcoming immunosuppressive mechanisms within the tumor microenvironment. Despite these advancements, challenges remain, including optimizing the treatment sequence, mitigating immune-related toxicities—particularly cytokine release syndrome (CRS)—and identifying predictive biomarkers to guide patient selection. As the role of monoclonal antibodies continues to expand, their integration into therapeutic regimens is transforming the management of FL, paving the way for chemotherapy-free treatment approaches and long-term disease control. This review provides an updated overview of mAbs therapies for FL, emphasizing the advances brought by BsAbs and ADCs toward more tailored and effective treatments. Full article

mRNA vaccines have demonstrated considerable efficacy and safety against SARS-CoV-2, limiting the pandemic burden worldwide. The emergence of new variants of concern and the decline in neutralizing activity observed several weeks post-vaccination reinforced the call for repeated mRNA vaccination. We and others have shown that vaccine efficacy does not exclusively rely on antibody neutralizing activites; Fc-effector functions play an important role as well. However, it is well known that long-term exposure and repeated antigen stimulation elicit the IgG4 subclass of antibodies, which are inefficient at mediating Fc-effector functions. In this regard, recent studies highlighted concerns about IgG4 induction by mRNA vaccines. Here, we explored the impact of repeated mRNA vaccination on IgG4 induction and its impact on Fc-effector functions. We observed anti-Spike IgG4 elicitation after three doses of mRNA vaccine; the antibody levels further increased with additional doses. Vaccine-elicited IgG4 preferentially bound the ancestral D614G Spike. We also observed that Breakthrough Infection (BTI) after several doses of vaccine strongly increased IgG1 levels but had no impact on IgG4 levels, thereby improving Fc-effector functions. Finally, we observed that elderly donors vaccinated with Moderna mRNA vaccines elicited higher IgG4 levels and presented lower Fc-effector functions than donors vaccinated with the Pfizer mRNA vaccine. Altogether, our results highlight the importance of monitoring the IgG subclasses elicited by vaccination. Full article

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with high metastatic potential, poor prognosis, and the absence of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2). The lack of these receptors limits the standard treatments, such as hormone therapies and HER2-targeted antibodies like trastuzumab. These challenges highlight the critical need for novel therapeutic strategies. CD147, a transmembrane glycoprotein overexpressed in TNBC, promotes tumor progression, metastasis, and chemoresistance, making it a promising therapeutic target. This study evaluates the antibody-dependent cellular cytotoxicity (ADCC) of HuM6-1B9, a humanized anti-CD147 antibody, against MDA-MB-231 cells, a TNBC model. Methods: CFSE-labelled MDA-MB-231 cells were co-cultured with PBMCs as effector cells (E:T ratio 80:1) in the presence of HuM6-1B9 and incubated for 4 h. Cells were then collected and stained with PI, and CFSE+/PI+ dead target cells were analyzed by flow cytometry. Results: Co-culturing MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) in the presence of HuM6-1B9 demonstrated effective ADCC induction without direct cytotoxicity. HuM6-1B9 induced 54.01% cancer cell death via ADCC, significantly outperforming trastuzumab (26.14%) while sparing PBMCs. Conclusion: These findings support HuM6-1B9 as a prospective TNBC therapeutic and warrant further investigation into its clinical potential. Full article