The field of antibody therapeutics is rapidly growing, with over 210 antibodies currently approved or in regulatory review and ~ 1,250 antibodies in clinical development. Antibodies are highly versatile molecules that, with strategic design of their antigen-binding domain (Fab) and the domain responsible for mediating effector functions (Fc), can be used in a wide range of therapeutic indications. Building on many years of progress, the biopharmaceutical industry is now advancing innovative research and development by exploring new targets and new formats and using antibody engineering to fine-tune functions tailored to specific disease requirements. In addition to considering the target and the disease context, however, the unique features of each therapeutic antibody trigger a diverse set of Fc-mediated effector functions. To avoid unexpected results on safety and efficacy outcomes during the later stages of the development process, it is crucial to measure the impact of antibody design on Fc-mediated effector function early in the antibody development process. Given the breadth of effector functions antibodies can deploy and the close interplay between the antibody Fab and Fc functional domains, it is important to conduct a comprehensive evaluation of Fc-mediated functions using an array of antigen-specific biophysical and cell-mediated functional assays. Here, we review antibody and Fc receptor properties that influence Fc effector functions and discuss their implications on development of safe and efficacious antibody therapeutics.

The binding kinetics of an antibody for its target antigen represent key determinants of its biological function and success as a novel biotherapeutic. Defining these interactions and kinetics is critical for understanding the pharmacological and pharmacodynamic profiles of antibodies in therapeutic applications, with line of sight to clinical translation. In this review, we discuss the latest developments in approaches to measure and modulate antibody-antigen interactions, including antibody engineering, novel antibody formats, current, and emerging technologies for measuring antibody-antigen binding interactions, and emerging perspectives within the field. We also explore how emerging computational methods are set to become powerful tools for modeling antibody-binding interactions under physiologically relevant conditions. Finally, we consider the therapeutic implications of modulating target engagement in terms of pharmacodynamics and pharmacokinetics.

FcγR (Fc gamma receptor) is a glycoprotein involved in various biological activities, such as inflammation and tumor immunity, and new ideas about the role of FcγR have also been published recently. Our study utilized journals derived from those published from 2004 to 2024 to analyze the research hotspots and cutting-edge ideas in this field.

All publications were searched using the web of science core collection database. VOSviewer, the R package Biblioshiny in R-studio and CiteSpace (version 6.1.R6) were utilized to perform bibliometric analysis which focused on authors, countries, organizations, keywords, etc.

The analysis of this article is based on 6849 articles related to FcγR from 107 countries and 37,487. The most cited reference in FcγR field is the article "Fcgamma Receptors as Regulators of Immune Responses," authored by Nimmerjahn. Country/region analysis shows that the United States of America (USA) has far more citation frequency and publications than other countries. The most recent hotspots and keywords are "COVID-19" and "SARS-CoV-2."

Through bibliometric analysis, we can clearly recognize the evolution of the field of FcγR research, from the original cellular immunity to tumor immunity to the occurrence of the latest viral immunity, which may guide the direction of research in the field and allow researchers to be more aware of the current status and frontiers of the field.

Microplastic particles (MP) ubiquitously occur in all environmental compartments where they interact with biomolecules, forming an eco-corona on their surfaces. The eco-corona affects the surface properties of MP and consequently how they interact with cells. Proteins, an integral component within the eco-corona, may serve as a ligand driving the interaction of MP with membrane receptors. To date, it is not known, whether eco-coronae originating from different environmental media differ in their proteinaceous compositions and whether these particles interact differently with cells. We show that the protein composition of the eco-coronae formed in freshwater (FW) and salt water (SW) are distinct from each other. We did not observe different adhesion strengths between MP coated with different eco-coronae and cells. However, the internalization efficiency and the underlying internalization mechanisms significantly differed between FW- and SW eco-coronae. By inhibiting actin-driven and receptor-mediated internalization processes using Cytochalasin-D, Amiloride, and Amantadine, we show that FW microplastic particles predominantly become internalized via phagocytosis, while macropinocytosis is more important for SW microplastic particles. Overall, our findings show that the origin of eco-coronae coatings are important factors for the cellular internalization of microplastic particles. This highlights the relevance of eco-coronae for adverse effects of environmentally relevant microplastic particles on cells and organisms.

Currently, porcine contagious pleuropneumonia (PCP) caused by Actinobacillus pleuropneumoniae (APP), poses a significant threat to the pig breeding industry. There is an urgent need for effective therapeutic and prophylactic treatments, especially those that can overcome the limitations associated with vaccines and antibiotics. This includes the development of novel antitoxin agents, immunomodulatory therapies, and alternative strategies like phage therapy and herbal extracts. Our previous study has demonstrated membrane protein caveolin-1 (CAV1) is a key protein that acts as a functional receptor of APP ApxI toxin by binding to its acylated region. Here, we developed recombinant human N-CAV1-Fc fusion protein and C-CAV1-Fc fusion protein. Both fusion proteins could tightly bind to ApxI toxin. N-CAV1-Fc and C-CAV1-Fc fusion proteins efficiently blocked the interaction between ApxI toxin and immortalized porcine alveolar macrophages (iPAMs), thereby inhibiting cell apoptosis caused by APP ApxI toxin. Furthermore, prophylactic and therapeutic CAV1-Fc treatments effectively protected mice from ApxI toxin-induced damage, as determined by reduced weight loss, apoptosis factor transcription, and pathological changes in the lungs. The protective effects of N-CAV1-Fc and C-CAV1-Fc showed clear dose-dependent efficacy in vivo. Protein kinetics data indicated that N-CAV1-Fc has a relatively longer half-life in vivo compared to C-CAV1-Fc, making it an excellent candidate for prevention and treatment of APP infections.

A man living with HIV was found to lack expression of CD16A on his natural killer (NK) cells and monocytes. Genetic analysis revealed compound heterozygous deletion of FCGR3A, the gene encoding CD16A. The case's NK cells showed: (a) no antibody-dependent cell-mediated cytotoxicity and very low spontaneous cytotoxicity; (b) an immature phenotype marked by high expression of CD94, CD2, NKG2A, and NKG2D, and low expression of KIR2DL2 and CD57; (c) no expression of KIR3DL1 and very low expression of FcRγ; and (d) normal cytokine production. The case's monocytes and DCs were similar phenotypically and functionally to those from the donors matched for HIV status, age, and percentage of NK cells in the peripheral blood. In contrast to previously reported people with CD16A deficiency, this man did not have a history of severe infections with herpes viruses, suggesting that other immune cells and/or immunoregulatory function of NK cells may compensate for deficiency of cytolytic NK cells.

Acinetobacter baumannii is a multidrug-resistant (MDR) nosocomial pathogen with high mortality rates, necessitating alternative preventive strategies. The RNA chaperone Hfq plays a key role in bacterial virulence, and its deletion attenuates pathogenicity. In this study, we evaluated an hfq deletion mutant (Δhfq) of A. baumannii ATCC 17978 as a live-attenuated vaccine candidate. Mice vaccinated intraperitoneally with 2.3 × 107 CFU of Δhfq, followed by a booster on day 28, exhibited 90 % survival after an intraperitoneal lethal challenge with MDR A. baumannii AB5075, whereas all unvaccinated controls succumbed. Vaccinated mice showed a significant reduction in bacterial burden in key organs (p < 0.001), sustained IgG levels, and an enhanced cytokine response with early IFN-γ and TNF-α activation, followed by a controlled IL-10 response. Serum pentraxin-3 (PTX3) levels were significantly lower in vaccinated mice post-challenge, correlating with reduced systemic inflammation and sepsis risk. Histopathological analysis revealed intact organ architecture in vaccinated mice, while unvaccinated mice displayed severe pathology. Functional immunogenicity assays demonstrated that serum from vaccinated mice enhanced neutrophil-mediated bacterial killing, and passive transfer of heat-inactivated serum conferred complete protection, confirming antibody-mediated immunity independent of complement activation. These findings highlight Δhfq as a promising live-attenuated vaccine that induces strong humoral and cellular immunity, preventing bacterial dissemination and lethal infection. Further studies are needed to elucidate its protective mechanisms and clinical potential.

Escherichia coli is a powerful and cost-effective platform for producing recombinant proteins. However, E. coli- produced proteins lack side-chain glycosylation and may be misfolded due to non-native disulfide bonds, often leading to poor immunogenicity. As a result, they are commonly perceived as unsuitable for use as antiviral vaccine antigens. This study addresses this challenge using the small 12 kDa envelope protein domain III of the Japanese encephalitis virus (JEV-EDIII) as a model. We demonstrate that the low immunogenicity of E. coli- produced proteins can be effectively overcome by employing a solubility-controlling peptide tag (SCP-tag) composed of five isoleucines (C5I). E. coli-produced JEV-EDIII oligomerized into 100 nm (Rh) soluble oligomers upon attachment of the C5I-tag, whereas the untagged JEV-EDIII remained monomeric (Rh ∼ 1.9 nm). The C5I-tag significantly enhanced anti-JEV EDIII IgG titers, as evidenced by ELISA, and increased the population of memory T cells in the spleen, as assessed by flow cytometry. Most notably, the C5I-tagged JEV-EDIII elicited neutralizing antibodies, confirmed by the FRNT50 neutralization assay using live JEV. These findings suggest that oligomerization via SCP-tagging offers a promising, adjuvant-free approach for producing neutralizing antibodies with long-term T cell memory, paving the way for developing E. coli- produced, protein domain-based vaccines.

Human immunoglobulin, delivered either intravenously (IVIg) or subcutaneously, is used to treat a range of immune-mediated neurological disorders. It has a role in acute or subacute inflammatory disease control and as a maintenance therapy in chronic disease management. This review considers mechanisms of IVIg action and the evidence for IVIg in neurological conditions. We use Guillain-Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) as frameworks to demonstrate an approach to IVIg use in acute and chronic dysimmune neurological conditions across two different healthcare systems: the UK and Australia. We highlight the benefits and limitations of IVIg and focus on practical considerations such as informed consent, managing risks and adverse effects, optimal dosing and monitoring response. We use these basic clinical practice principles to discuss the judicious use of an expensive and scarce blood product with international relevance.

Diabetic retinopathy (DR), one of the most common severe complications of diabetes, has become a leading cause of blindness among the working population without a fundamental treatment. Proliferative DR (PDR) is the advanced stage of DR. Recent studies have shown that inflammation is closely related to PDR, as it promotes leukocyte adhesion, breakdown of the blood-retinal barrier, and pathological neovascularization, but the key regulatory genes involved remained unclear. We aim to identify inflammation-related biomarkers in PDR.

We downloaded and merged PDR-related datasets GSE102485, GSE94019, and GSE60436, comprising a total of 13 control samples and 37 samples from PDR patients, and conducted a joint analysis of inflammation-related genes (IRGs). Differential analysis, functional enrichment analysis, WGCNA and LASSO were used to identify key genes and their functions in the pathogenesis of PDR. Dataset GSE241239, which contains retinal sequencing data from mice, was used for external validation. Additionally, single-cell RNA analysis using GSE165784, which includes five human-derived PDR samples, was conducted to investigate the cellular expression of Fc Gamma Receptor IA (FCGR1A) and Integrin Subunit Alpha L (ITGAL). Finally, the expression of FCGR1A and ITGAL was validated in DR mouse models and high glucose-induced cell models.

Nine key genes associated with the pathogenesis of PDR were identified. Further screening identified FCGR1A and ITGAL as potential therapeutic targets, with single-cell analysis showing their primary distribution in microglia. In vivo and in vitro experiments confirmed localization of FCGR1A and ITGAL in microglia and significant elevation within DR mouse models.

Comprehensive analysis indicates, for the first time, that FCGR1A and ITGAL are key inflammation-related genes involved in the pathogenesis of PDR mediated by microglia. FCGR1A and ITGAL are promising therapeutic targets for PDR.

The tumor microenvironment (TME) plays a critical role in ovarian cancer (OC) progression, yet the relationship between immune and stromal scores within the TME and prognostic outcomes remains poorly understood. Immune and stromal cell scores were computed using the "estimate" R package, which enabled the assessment of immune and stromal components in OC samples. We then performed univariate and multivariate Cox regression analyses to identify prognostic factors associated with these scores using data from The Cancer Genome Atlas (TCGA). Additionally, LASSO Cox regression were employed to identify key prognostic genes linked to immune infiltration. Our analysis of OC expression data identified 1,667 differentially expressed genes (DEGs) associated with immune and stromal scores. From these, we developed a 6-gene risk model, consisting of ALOX5AP, FCGR1C, GBP2, IL21R, KLRB1, and PIK3CG, which effectively stratified OC patients into high-risk and low-risk groups. Survival analysis and area under the curve (AUC) assessment confirmed the model's strong predictive accuracy. Furthermore, drug sensitivity predictions indicated that sorafenib was particularly effective in high-risk patients, with this finding validated through in vitro experiments. The 6-gene TME-related risk model offers robust prognostic capabilities for OC and could serve as a valuable tool for clinical stratification and personalized treatment approaches.

Glioma is the most common primary malignant intracranial tumor with limited treatment options and a dismal prognosis. This study aimed to develop a robust gene expression-based prognostic signature for GBM using the Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) datasets. Using WGCNA and LASSO algorithms, we identified four MHC-related genes (TNFSF14, MXRA5, FCGR2B, and TNFRSF9) as prognostic biomarkers for glioma. A risk model based on these genes effectively stratified patients into high- and low-risk groups with distinct survival outcomes across TCGA and CGGA cohorts. This signature correlated with immune pathways and glioma progression mechanisms, showing strong associations with immune function and tumor microenvironment infiltration patterns. The risk score reflected tumor microenvironment remodeling, suggesting its prognostic relevance. We further propose I-BET-762 and Enzastaurin as potential therapeutic candidates for glioma. In conclusion, the four-gene signature we identified and the corresponding risk score model constructed from it provide valuable tools for the prognosis prediction of glioblastoma multiforme (GBM) and may guide personalized treatment strategies. The least absolute shrinkage and selection operator (LASSO) risk score has demonstrated significant prognostic evaluation utility in clinical GBM patients, bringing potential implications for patient stratification and the optimization of treatment regimens.

Bruton's tyrosine kinase (BTK) is a therapeutically validated drug target. Small-molecule inhibitors of BTK have changed the treatment paradigms of multiple B-cell malignancies and evolved over three generations to overcome clinical challenges. Four drugs are now approved by the FDA, including the first-in-class drug ibrutinib and successively approved acalabrutinib, zanubrutinib, and pirtobrutinib. The third-generation drug pirtobrutinib, which binds non-covalently to BTK, is expected to overcome resistance mutations at the covalent binding Cys481 residue of the first and second-generation drugs that covalently bind to BTK. However, some newly identified non-Cys481 resistance mutations to pirtobrutinib have shown their co-resistance to some of the covalent inhibitors, and this leaves a major unmet need that is promoting the development of next-generation BTK-targeted therapeutics. More non-covalent BTK inhibitors with differentiated binding modes are under development, and the ongoing development focus of next-generation therapeutics involves new and alternative directions to target BTK using dual-binding inhibitors and degraders of BTK, as well as its allosteric inhibitors. Recent exploration of the differentiated features of BTK inhibitors in various aspects has shown the possible link between their different features and different functional and therapeutic consequences. This review summarizes the key differentiated features of the BTK inhibitors approved by the FDA and others under development to add knowledge for their therapeutic application and future development. Long-term follow-up updates of clinical outcomes of the earlier developed drugs are also included, together with direct and indirect comparisons of efficacy and safety between the different generations of drugs. The ongoing development status of next-generation BTK-targeted therapeutics is described, with a discussion on their therapeutic potential and some limitations.

Targeted tumor delivery may be required to potentiate the clinical benefit of innate immune modulators. The objective of the study was to apply an antibody-drug conjugate (ADC) approach to STING agonism and develop a clinical candidate.

XMT-2056, a HER2-directed STING agonist ADC, was designed, synthesized, and tested in pharmacology and toxicology studies. The ADC was compared with a clinical benchmark intravenously administered a STING agonist.

XMT-2056 achieved tumor-targeted delivery of the STING agonist upon systemic administration in mice and induced innate antitumor immune responses; single dose administration of XMT-2056 induced tumor regression in a variety of tumor models with high and low HER2 expressions. Notably, XMT-2056 demonstrated superior efficacy and reduced systemic inflammation compared with a free STING agonist. XMT-2056 exhibited concomitant immune-mediated killing of HER2-negative cells specifically in the presence of HER2-positive cancer cells, supporting the potential for activity against tumors with heterogeneous HER2 expression. The antibody does not compete for binding with trastuzumab or pertuzumab, and a benefit was observed when combining XMT-2056 with each of these therapies as well as with trastuzumab deruxtecan ADC. The combination of XMT-2056 with anti-PD-1 conferred benefit on antitumor activity and induced immunologic memory. XMT-2056 was well tolerated in nonclinical toxicology studies.

These data provide a robust preclinical characterization of XMT-2056 and provide rationale and strategy for its clinical evaluation.

Efficient antibody responses are crucial for combating infectious diseases and vaccination remains a cornerstone of this effort. This study introduces a novel approach for enhancing immune responses in wild-type mice by utilizing pre-formed immune complexes, using the receptor-binding domain (RBD) of SARS-CoV-2 as a model antigen to illustrate the broader potential of the concept. Specifically, we found that pre-treating the antigen with bis-maleimide, a chemical linker that facilitates protein cross-linking, significantly enhances antibody production. Moreover, in vitro cross-linking of antigen to unrelated IgG using bis-maleimide generated immune complexes that markedly enhanced antigen-specific antibody responses, likely by mimicking natural memory-like mechanisms, suggesting that bis-maleimide pre-treated antigens may similarly engage IgG in vivo. In contrast, antigen crosslinking with IgA or IgM did not yield comparable effects, highlighting the unique capacity of IgG to boost immunogenicity. By leveraging the principles of immune memory, this study demonstrates the potential of pre-formed immune complexes to significantly enhance vaccine efficacy using an antigen-independent strategy broadly applicable to diverse pathogens.

The phagocytosis of macrophages to tumor cells represents an alluring strategy for cancer immunotherapy; however, its effectiveness is largely hindered by the detrimental upregulation of anti-phagocytic signals and insufficient expression of pro-phagocytic signals of tumor cells. Here, a pro-phagocytic polymer-based nanocomplex is designed to promote the macrophage engulfment of tumor cells through concurrent modulation of both the "eat me" and "don't eat me" signals. The nanocomplex MNCCD47i-CALRt is formed by complexing a synthetic PAMAM derivative (G4P-C7A) that is capable of intrinsically inducing the exposure of calreticulin (CALR, a crucial pro-phagocytic protein) and a small inference RNA that can inhibit the expression of CD47 (a primary anti-phagocytic protein). MNCCD47i-CALRt can significantly delay tumor growth and prolong the survival of tumor-bearing mice with negligible hematopoietic toxicity in multiple murine colorectal cancer models. Furthermore, the pro-phagocytic capacity of MNCCD47i-CALRt is validated in the patient-derived tumor organoid model. Collectively, the phagocytosis-promoting nanocomplex provides a simple and potent strategy for boosting macrophage-mediated cancer immunotherapy.

Adaptive immunity after a stroke results in a shift of T cells between compartments, leading to peripheral lymphopenia and an increased number of T cells within the brain lesion. Stroke-associated infection (SAI) presents a clinically significant challenge in stroke units. The role of T-cell subsets in the post-stroke immune response and in SAI remains unclear. Thus, we aimed to observe the quantitative changes of circulating CD4+, CD8+, double-negative T cells, and the CD4+/CD8+ ratio in stroke and SAI.

We prospectively assessed circulating CD4+, CD8+, and double-negative T cells using flow cytometry in 52 patients on days 1, 3, 10, and 90 after ischemic stroke. We compared the results to those obtained from age-, sex-, and vascular risk factor-matched controls. We analyzed lymphocyte parameters in relation to clinical outcome, SAI, infarct lesion volume, and risk factor burden.

There were no differences in the studied parameters between stroke patients and controls, as well as between subjects with and without SAI. A higher percentage of CD4+ T cells and a higher CD4+/CD8+ ratio correlated with better clinical status in the acute and subacute phases, while CD8+ T cells showed the opposite correlation. The percentage of CD8+ T cells positively correlated with CRP levels during the acute and subacute phases of stroke, as well as in the control group. A negative correlation was noted between the percentage of CD4+ T cells on D1 and the serum CRP level on D10 after stroke. Similarly, the CD4+/CD8+ ratio on D1 negatively correlated with CRP on D1, D3, and D10. In patients with a history of hypertension (HT), there was a higher percentage of CD8+ T cells and a lower percentage of CD4+ T cells in the acute phase of stroke than those without HT.

Receptors for FcγR on human neutrophils constitute an important mechanism for the recognition of opsonized microorganisms and for cell activation. Human neutrophils express 2 FcγR: FcγRIIa and FcγRIIIb. Previously, it has been reported that activation of each FcγR induces different neutrophil responses by triggering distinct signal transduction pathways, although what particular signal transduction pathway is triggered by each FcγR has not been completely elucidated. It has also been reported that PKC is important for FcγR signaling and that each FcγR may activate different PKC isoforms. Therefore, we explored whether FcγRIIa or FcγRIIIb activates different PKC isoforms in human neutrophils and whether activation of these PKC isoforms results in different neutrophil responses. Hence, either FcγRIIa or FcγRIIIb was selectively cross-linked by monoclonal antibodies in the presence or absence of pharmacological inhibitors for various PKC isoforms. Inhibition of PKCα or PKCδ blocked FcγRIIa-induced reactive oxygen species productions. In contrast, inhibition of PKCα and/or PKCβ blocked FcγRIIIb-induced reactive oxygen species production. Also, inhibition of all PKC isoforms did not affect the FcγRIIa-induced increase in intracellular calcium concentration ([Ca2+]i), while inhibition of PKCα blocked FcγRIIIb-induced increase in [Ca2+]i. Additionally, inhibition of PKCδ blocked FcγRIIa-induced ERK phosphorylation, while inhibition of PKCα prevented FcγRIIIb-induced ERK phosphorylation. These results suggest that both FcγRIIa and FcγRIIIb activate unique PKC isoforms and that activation of these PKC isoforms can selectively regulate different neutrophil functions. These findings also reinforce the idea that each FcγR in human neutrophils triggers distinct signal transduction pathways.

Mounting evidence shows that gut microbiota communities and the human immune system coexist and influence each other, and there are a number of reports of a correlation between specific changes in gut microbiota and the occurrence of autoimmune diseases. B lymphocytes play a central role in the regulation of both gut microbiota communities and in autoimmune diseases. Here, we summarize evidence of the influence of gut microbiota-B cell pathways on autoimmune diseases and how B cells regulate microorganisms, which provides mechanistic insights with relevance for identification of potential therapeutic targets and related fields.

Fibrinogen-like (Fgl2) protein belongs to fibrinogen super family, which catalyzes the conversion of prothrombin to thrombin and is involved in the coagulation process. There are two different forms of functional Fgl2 protein: membrane associated Fgl2 (mFgl2) and soluble Fgl2 (sFgl2). mFgl2, as a type II transmembrane protein with property with prothrombinase activity from its N-terminal fragment, was extensively secreted or expressed by inflammatory macrophages, dendritic cells (DCs), Th1 cells and endothelial cells. While sFgl2 was mainly produced by regulatory T cells (Tregs) and then secreted into the vasculature, which contributes to autoimmune disease by regulating maturation of (DCs), polarization of macrophage, inhibiting T cell proliferation and differentiation and inducing apoptosis of B cells. In particular, emerging evidence has shown that Fgl2 is implicated in female reproductive system that contributes to embryo development, ovarian granulosa cells differentiation and implantation failure. This article summarizes the role and potential mechanisms of Fgl2 in reproduction and identifies research gaps along with the future directions.

Immune memory is influenced by the frequency and type of antigenic challenges. Here, we performed a cross-sectional comparison of immune parameters following a BA.1 breakthrough infection in individuals with prior hybrid immunity (conferred by infection and vaccination) versus those solely vaccinated in a cohort of health care workers in Lyon, France. The results showed higher levels of serum anti-receptor binding domain (RBD) antibodies and neutralizing antibodies against BA.1 post-infection in the vaccine-only group. Individuals in this group also showed a decrease in memory B cells against the ancestral strain but an increase in those specific and cross-reactive to BA.1, suggesting a more limited immune imprinting. Conversely, hybrid immunity prevents the decrease in antibody dependent cellular cytotoxicity (ADCC) response, possibly by limiting IgG4 class-switching and enhanced anti-N responses post-infection. This highlights that BA.1 breakthrough infection induces different immune responses depending on prior history of vaccination and infection, which should be considered for further vaccination guidelines.

Sheep red blood cells (SRBC) has a long history as a classical T-cell dependent (TD) antigen. Due to its cost-effectiveness, easy accessibility, and ability to elicit a robust antibody immune response, SRBC continues to be widely used in studies related with humoral immunity modulation, vaccine development, and immunoactivity/immunotoxicity testing of bioactive agents. However, detecting the relative affinity levels of SRBC-specific antibodies in SRBC-immunized animal models remains challenging. Using flow cytometry, we established a detection system capable of quickly and accurately assessing the SRBC-specific antibody relative affinity levels in humoral samples (e.g., serum, tissue fluid) of SRBC-immunized mouse models. We further validated this method using affinity maturation-deficient mice, demonstrating that this method can distinguish affinity levels of the antibodies from different samples. This approach is simple and efficient, providing an accurate and effective technological solution for research on mechanisms of humoral immunity, antibody affinity maturation, vaccine response, and immunoactivity/immunotoxicity testing.

Ovarian cancer is the most lethal gynaecological cancer and treatment options remain limited. In a recent first-in-class Phase I trial, the monoclonal IgE antibody MOv18, specific for the tumour-associated antigen Folate Receptor-α, was well-tolerated and preliminary anti-tumoural activity observed. Pre-clinical studies identified macrophages as mediators of tumour restriction and pro-inflammatory activation by IgE. However, the mechanisms of IgE-mediated modulation of macrophages and downstream tumour immunity in human cancer remain unclear. Here we study macrophages from patients with epithelial ovarian cancers naive to IgE therapy. High-dimensional flow cytometry and RNA-seq demonstrate immunosuppressive, FcεR-expressing macrophage phenotypes. Ex vivo co-cultures and RNA-seq interaction analyses reveal immunosuppressive associations between patient-derived macrophages and regulatory T (Treg) cells. MOv18 IgE-engaged patient-derived macrophages undergo pro-inflammatory repolarisation ex vivo and display induction of a hyperinflammatory, T cell-stimulatory subset. IgE reverses macrophage-promoted Treg cell induction to increase CD8+ T cell expansion, a signature associated with improved patient prognosis. On-treatment tumours from the MOv18 IgE Phase I trial show evidence of this IgE-driven immune signature, with increased CD68+ and CD3+ cell infiltration. We demonstrate that IgE induces hyperinflammatory repolarised states of patient-derived macrophages to inhibit Treg cell immunosuppression. These processes may collectively promote immune activation in ovarian cancer patients receiving IgE therapy.

Microglia and macrophages are critical mediators of immune responses in the central nervous system. Their roles range from homeostatic maintenance to the pathogenesis of autoimmune demyelinating diseases such as multiple sclerosis and neuromyelitis optica spectrum disorder. This review explores the origins of microglia and macrophages, as well as their mechanisms of activation, interactions with other neural cells, and contributions to disease progression and repair processes. It also highlights the translational relevance of insights gained from animal models and the therapeutic potential of targeting microglial and macrophage activity in multiple sclerosis and neuromyelitis optica spectrum disorder.

Monoclonal antibodies have two core mechanisms of protection: an antibody's antigen-binding fragment (Fab) can bind and neutralize viral pathogens and its fragment crystallizable domain (Fc) catalyzes effector functions. We investigated the relative contribution of Fab- versus Fc-mediated mechanisms of protection through passive administration of distinct forms of the pan-reactive anti-influenza antibody CR9114. We demonstrated that the contribution of Fc-independent (Fab-dependent) versus Fc-dependent mechanisms of protection is defined by the route of administration. We used CR9114 variants (wild-type, two Fc-silenced variants, or the bivalent antigen-binding fragment F(ab')2), administered either intravenously or intranasally. We found that intravenously-administered CR9114 requires the Fc domain to provide potent, pre-exposure protection against influenza A and B viral challenge. In contrast, when CR9114 was administered locally to the nasal mucosa, the main mode of protection was provided by F(ab')2, and was largely Fc-independent. Importantly, this mode of protection following intranasal administration also applied to non-neutralized influenza B strains. Moreover, intranasal administration resulted in an increase in potency against influenza A/H1N1, A/H5N1, A/H3N2, B/Yam and B/Vic compared to intravenous administration up to 50-fold. These results shed new light on the application of monoclonal antibodies such as CR9114 to combat viral infection locally, and will help inform clinical strategies of pre-exposure prophylaxis. More fundamentally, this study uncovers distinct modes of protection for systemic versus intranasally-administered prophylactic antibodies.

Monoclonal antibodies have successfully been used for a variety of indications. Many therapeutic antibodies are IgG1 and elicit effector functions as part of their mechanism of action. It is well known that aggregate levels should be controlled for therapeutic antibodies. Although there are several reports describing the impact of antibody aggregates on FcγR binding, most of these have been performed with surface plasmon resonance in an avidity-based format. What is less well known is which Fcγ receptor is most impacted by antibody aggregation and how antibody aggregates impact binding to Fcγ receptors in solution-based formats and in cell-based assays.

An effector-competent IgG1 (mAb1) was forcibly degraded and fractionated by size exclusion chromatography to enrich for aggregates. The fractions were examined for FcγR binding by SPR with different formats and in solution. The fractions were also analyzed with cell-based FcγR reporter assays.

All Fcγ receptors displayed increased binding to enriched mAb1 aggregates in the avidity-based SPR methods and in solution, with FcγRIIa impacted the most. When examined with an antibody-down SPR format that is not usually susceptible to avidity, FcγRIIa did not show increased binding with mAb1 aggregation. Although activity for mAb1 aggregates increased slightly in an FcγRIIa cell-based reporter assay, it decreased in the FcγRIIIa reporter assay (most likely due to differences in fucosylation from the reference standard).

Monoclonal antibody aggregation can impact FcγR binding for avidity-based binding formats. Even at low levels of antibody aggregation, FcγRII binding increases substantially.

Kam and colleagues discovered that FcγRIIb can specifically bind to Aβ42 oligomers (AβOs). The N-terminal residues F4 and D7 of Aβ42, as well as the W115 residue in domain D2 of FcγRIIb, are involved in this binding. However, the specificity of the FcγRIIb receptor's binding sites for AβOs and their dependence on different AβO species, including dimers (D/DT), trimers (T/TT), tetramers (Te/TeT), and pentamers (P/PT) during both the primary (P1) and secondary nucleation phases (P2), remains unknown. To address this, we employed molecular dynamics (MD) simulations to investigate the interactions between the extracellular domains D1 and D2 (FDD) of FcγRIIb and AβOs of varying sizes in the two different phases. We discovered that three specific fragments (f1, f2, and f3) of domain D2 in FDD are the primary binding sites for AβO species. Furthermore, among AβOs of the same molecular weight, those from the P2 phase exhibit a stronger binding affinity for FDD than those from the P1 phase. The distinction is ascribed to the stronger dependence on the hydrophobic residues in the β1 and β2 regions for the binding of AβOs in P2 (including TT, TeT, and PT) than that (including D, Te, and P) in the P1 phase. In the P1 phase, these AβOs prefer to achieve binding to FDD through their N-terminal residues; however, by this, we identified that the species observed in Kam's experiment to bind FcγRIIb should probably be the tetrameric AβO (Te) in the P1 phase. Moreover, within both the P1 and P2 phases, we predicted that the trimeric AβO species in either the P1 or P2 phase is the strongest binding ligand for the FcγRIIb receptor. This study provides a comprehensive molecular perspective on the interaction between FcγRIIb and AβO in P2, which is of significant importance for the development of therapeutic strategies targeting Alzheimer's disease (AD) and autoimmune diseases.

IgG antibodies, required for a functional immune system, recognize antigens and neutralize pathogens using their Fab regions, while signaling to the immune system by binding to host Fc γ receptors (FcγRs) through their Fc regions. These FcγR interactions initiate and modulate antibody-mediated effector functions that are essential for host immunity, therapeutic monoclonal antibody effectiveness and IgG-mediated pathologies. FcγRs include both activating and inhibitory receptors and the relative binding affinities of the IgG Fc region to FcγRs that generate opposing signals is a key determinant of the immune response. Substantial research effort has been devoted to understanding and manipulating FcγR interactions to decipher their fundamental biological activities and to develop therapeutic monoclonal antibodies with tailored effector functions. However, a common Fc-FcγR binding interface, the high sequence identity of FcγRs, and the inherent conformational dynamics of the IgG Fc region, have prohibited a full understanding of these interactions, even when employing state-of-the-art biophysical and biological methods. Here, we used site-saturation libraries of the human IgG1 Fc region to determine the effective affinities of more than 98% of all possible single-site amino acid substitutions in the Fc to all human FcγRs, as well as the most common FcγR polymorphisms. We provide a comprehensive analysis of Fc amino acid variations that determine Fc stability, orthosteric control of FcγR binding, and short- and long-range allosteric control of FcγR binding. We also predict the relative activating versus inhibitory effector function capacity of nearly every possible single-site Fc mutation.

Bruton's tyrosine kinase (BTK) inhibitors constitute a promising category of small molecules for the therapy of diverse B-cell malignancies and autoimmune disorders. This review examines the journey of BTK inhibitors from their discovery to clinical development, highlighting key milestones in their design, mechanism of action, and progression through preclinical and clinical stages. Initially identified through high-throughput screening of compound libraries, early BTK inhibitors were optimized for selectivity and potency. The discovery of ibrutinib, the first Food and Drug Administration (FDA)-approved BTK inhibitor, marked a significant breakthrough, providing a new therapeutic option for patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Following this success, numerous second-generation inhibitors have been identified to address resistance mechanisms, improve pharmacokinetics, and target specific patient populations. The challenges faced during the transition from preclinical validation to clinical trials have been discussed. Additionally, ongoing trials and emerging data on novel BTK inhibitors provide insights into their evolving role in oncology and immunology. This review emphasizes the importance of rational drug design and clinical strategy in shaping the future of BTK inhibitors.

The effectiveness of therapeutic antibodies is often associated with their Fc-mediated effector functions, such as antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. These functions rely on interactions between Fc gamma receptors (FcγRs) on immune cells and the Fc region of antibodies. Genetic variations in these receptors, known as FcγR polymorphisms, can influence therapeutic outcomes by altering receptor expression levels, affinity, and function. This review examines the impact of FcγR polymorphisms on antibody therapy, emphasizing their role in developing and optimizing functional bioassays to assess product quality. Understanding these polymorphisms is essential for refining bioassays, which are crucial for accurately characterizing antibody products and ensuring consistency in manufacturing processes.

Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disease characterized by reduced platelet counts due to immune system dysregulation caused by many factors, including genetics, autoimmune diseases, infections, and inflammations. Therefore, the current study aimed to evaluate immunological markers such as the expression level of lysosomal associated membrane protein 3 (LAMP-3), also known as CD63, and the expression level of Fc-gamma receptor I (FcγRI), also known as CD64 and also investigate the association of Fc-gamma receptor IIIA (FcγRIIIA) 158 V/F polymorphism to the risk of ITP.

A total of 180 subjects; 60 ITP patients, 60 patients with thrombocytopenia of other causes and 60 controls were enrolled into our study. The expression level of CD63 was done using reverse transcription quantitative PCR (RTqPCR), while CD64 expression level was done by flow cytometry. The polymorphism of FcγRIIIA 158 V/F gene was analyzed by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis. Finally, CD63 and CD64 protein-protein interactions were done by using the STRING online database.

The expression of CD63 was significantly elevated in ITP patients than thrombocytopenia patients and healthy control. Also there was high expression level of CD64 on granulocytes and monocytes from ITP patients than other groups. Receiver operating characteristic curve (ROC curve) analysis of CD63 showed an area under the curve (AUC) revealed of 1.00, sensitivity of 100% and specificity of 100%; while for CD64 on granulocytes, AUC of 0.998 as well as a sensitivity of 96.66% and specificity of 93.33%. Regarding FcγRIIIa 158 V/F polymorphism, all patients and healthy volunteers included in this study showed the wild FF genotype.

The expression of both CD63 and CD64 were significantly increased in ITP patients and could be good biomarkers to diagnose ITP. Additionally, there is no association between FcγRIIIa 158 V/F polymorphism and the risk of ITP disease.

Hypertension is the most frequent health-related condition worldwide and is a primary risk factor for renal and cardiovascular diseases. However, the underlying molecular mechanisms are still poorly understood. To uncover these mechanisms, multi-omics studies have significant potential, but such studies are challenged by genetic and environmental confounding - an issue that can be effectively reduced by studying intra-pair differences in twins. Here, we coupled data on hypertension diagnoses from the nationwide Danish Patient Registry to a study population of 740 twins for whom genome-wide DNA methylation and gene expression data were available together with measurements of systolic and diastolic blood pressure. We investigated five phenotypes: incident hypertension cases, systolic blood pressure, diastolic blood pressure, hypertension (140/90 mmHg), and hypertension (130/80 mmHg). Statistical analyses were performed using Cox (incident cases) or linear (remaining) regression analyses at both the individual-level and twin pair-level. Significant genes (p < 0.05) at both levels and in both types of biological data were investigated by bioinformatic analyses, including gene set enrichment analysis and interaction network analysis. Overall, most of the identified pathways related to the immune system, particularly inflammation, and biology of vascular smooth muscle cell. Of specific genes, lysine methyltransferase 2 A (KMT2A) was found to be central for incident hypertension, ataxia-telangiectasia mutated (ATM) for systolic blood pressure, and beta-actin (ACTB) for diastolic blood pressure. Noteworthy, lysine methyltransferase 2A (KMT2A) was also identified in the systolic and diastolic blood pressure analyses. Here, we present novel biomarkers for hypertension. This study design is surprisingly rare in the field of hypertension. We identified biological pathways related to vascular smooth muscle cells and the immune system, particular inflammation, to be associated with hypertension and blood pressure. Of specific genes, we identified KMT2A (lysine methyltransferase 2A) to be central for blood pressure and hypertension development. ACTB beta-actin, ATM ataxiatelangiectasia mutated, BP blood pressure, EWAS epigenome-wide association studies, KMT2A lysine methyltransferase 2A, LMER linear mixed effect regression, LR linear regression, TWAS transcriptome-wide association studies.

B cells contribute to the development of pain after sciatic nerve chronic constriction injury (CCI) via binding of immunoglobulin G (IgG) to Fc gamma receptors (FcγRs) in the lumbar dorsal root ganglia (DRG) and spinal cord. Yet the contribution of B cells to pain after different types of peripheral nerve injury is uncertain. Using male and female mice, we demonstrate a divergent role for B cell-IgG-FcγR signaling underlying mechanical allodynia between CCI, nerve crush (NC), spared nerve injury (SNI), and spinal nerve ligation (SNL). Depletion (monoclonal anti-CD20) or genetic deletion (muMT mice) of B cells prevented development of allodynia following NC and CCI, but not SNI or SNL. In apparent contradiction, circulating levels of autoreactive IgG and circulating immune complexes were increased in all models, though more prominent following NC and CCI. Passive transfer of IgG from SNI donor mice induced allodynia in CCI muMT recipient mice, demonstrating that IgG secreted after SNI is pronociceptive. To investigate why pronociceptive IgG did not contribute to mechanical allodynia after SNI, we evaluated levels of the Fc receptor γ subunit. SNI or SNL did not increase γ subunit levels in the DRG and spinal cord, whereas CCI and NC did, in agreement with B cell-dependent allodynia in these models. Together, the results suggest that traumatic peripheral nerve injury drives secretion of autoreactive IgG from B cells. However, levels of cognate FcγRs are increased following sciatic nerve constriction and crush, but not transection, to differentially regulate pain through the B cell-IgG-FcγR axis.

Immunoglobulin G (IgG) antibodies are an essential component of humoral immunity protecting the host from recurrent infections. Among all antibody isotypes, IgG antibodies have a uniquely long half-life, can basically reach any tissue in the body, and have the ability to kill opsonized target cells, which has made them the molecule of choice for therapeutic interventions in cancer and autoimmunity. Moreover, IgG antibodies in the form of pooled serum IgG preparations from healthy donors are used to treat chronic inflammatory and autoimmune diseases, providing evidence that serum IgG antibodies can have an active immunomodulatory activity. Research over the last two decades has established that the single sugar moiety attached to each IgG heavy chain plays a very important role in modulating the pro- and anti-inflammatory activities of IgG. Moreover, specific sugar moieties such as sialic acid and galactose residues can serve as highly specific biomarkers for ongoing inflammatory processes. This chapter will summarize how different sugar residues in the IgG sugar moiety change upon inflammation and how such changes may translate to altered IgG function and hence maybe useful for optimizing or modulating the function of therapeutic antibodies.

Haemophilus ducreyi causes the genital ulcer disease chancroid and cutaneous ulcers in children. To study its pathogenesis, we developed a human challenge model in which we infect the skin on the upper arm of human volunteers with H. ducreyi to the pustular stage of disease. The model has been used to define lesional architecture, describe the immune infiltrate into the infected sites using flow cytometry, and explore the molecular basis of the immune response using bulk RNA-seq. Here, we used single cell RNA-seq (scRNA-seq) and spatial transcriptomics to simultaneously characterize multiple cell types within infected human skin and determine the cellular origin of differentially expressed transcripts that we had previously identified by bulk RNA-seq. We obtained paired biopsies of pustules and wounded (mock infected) sites from five volunteers for scRNA-seq. We identified 13 major cell types, including T- and NK-like cells, macrophages, dendritic cells, as well as other cell types typically found in the skin. Immune cell types were enriched in pustules, and some subtypes within the major cell types were exclusive to pustules. Sufficient tissue specimens for spatial transcriptomics were available from four of the volunteers. T- and NK-like cells were highly associated with multiple antigen presentation cell types. In pustules, type I interferon stimulation was high in areas that were high in antigen presentation-especially in macrophages near the abscess-compared to wounds. Together, our data provide a high-resolution view of the cellular immune response to the infection of the skin with a human pathogen.IMPORTANCEA high-resolution view of the immune infiltrate due to infection with an extracellular bacterial pathogen in human skin has not yet been defined. Here, we used the human skin pathogen Haemophilus ducreyi in a human challenge model to identify on a single cell level the types of cells that are present in volunteers who fail to spontaneously clear infection and form pustules. We identified 13 major cell types. Immune cells and immune-activated stromal cells were enriched in pustules compared to wounded (mock infected) sites. Pustules formed despite the expression of multiple pro-inflammatory cytokines, such as IL-1β and type I interferon. Interferon stimulation was most evident in macrophages, which were proximal to the abscess. The pro-inflammatory response within the pustule may be tempered by regulatory T cells and cells that express indoleamine 2,3-dioxygenase, leading to failure of the immune system to clear H. ducreyi.

Effective delivery of lipid nanoparticles (LNPs) and their organ- or cell-type targeting are paramount for therapeutic success. Achieving this requires a comprehensive understanding of protein corona dynamics and the identification of cell receptors involved in the recognition and uptake of LNPs. We introduce a simple, fast, and in situ strategy by a biosensor-based "Fishing" method to uncover protein corona formation on LNPs and identify key receptors of human blood cells that are responsible for the recognition and binding of human plasma corona on the surface of LNPs. Unexpectedly, we observed a significant presence of immunoglobulins with high abundance, especially anti-PEG antibodies, within the LNP corona. These antibodies, along with complement opsonization, drive colony-stimulating factor 2 receptor β (CSF2RB)-mediated phagocytosis by human myeloid cells. These compositions of the human plasma corona and their interactions with neighboring proteins are critical for the recognition and binding of LNPs by cell receptors and cellular uptake. Our findings highlight the pivotal role of anti-PEG antibodies in the circulation and phagocytosis of LNPs in vivo. This approach offers profound insights into nanomaterial behavior in vivo, paving the way for the enhanced design and efficacy of LNP-based therapies.