Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

Immune cell phenotyping frequently detects lineage-unrelated receptors. Here, we report that surface receptors can be transferred from primary macrophages to CD4 T cells and identify the Fcγ receptor CD32 as driver and cargo of this trogocytotic transfer. Filamentous CD32+ nanoprotrusions deposit distinct plasma membrane patches onto target T cells. Transferred receptors confer cell migration and adhesion properties, and macrophage-derived membrane patches render resting CD4 T cells susceptible to infection by serving as hotspots for HIV-1 binding. Antibodies that recognize T cell epitopes enhance CD32-mediated trogocytosis. Such autoreactive anti-HIV-1 envelope antibodies can be found in the blood of HIV-1 patients and, consistently, the percentage of CD32+ CD4 T cells is increased in their blood. This CD32-mediated, antigen-independent cell communication mode transiently expands the receptor repertoire and functionality of immune cells. HIV-1 hijacks this mechanism by triggering the generation of trogocytosis-promoting autoantibodies to gain access to immune cells critical to its persistence.

Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC.

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this "affinity profiling" approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

Immunoglobulins and serum proteins impair anti-tumor NK cell effector functions in malignant ascites.

Introduction: Malignant ascites indicates ovarian cancer progression and predicts poor clinical outcome. Various ascites components induce an immunosuppressive crosstalk between tumor and immune cells, which is poorly understood. In our previous study, imbalanced electrolytes, particularly high sodium content in malignant ascites, have been identified as a main immunosuppressive mechanism that impaired NK and T-cell activity. Methods: In the present study, we explored the role of high concentrations of ascites proteins and immunoglobulins on antitumoral NK effector functions. To this end, a coculture system consisting of healthy donor NK cells and ovarian cancer cells was used. The anti-EGFR antibody Cetuximab was added to induce antibody-dependent cellular cytotoxicity (ADCC). NK activity was assessed in the presence of different patient ascites samples and immunoglobulins that were isolated from ascites. Results: Overall high protein concentration in ascites impaired NK cell degranulation, conjugation to tumor cells, and intracellular calcium signaling. Immunoglobulins isolated from ascites samples competitively interfered with NK ADCC and inhibited the conjugation to target cells. Furthermore, downregulation of regulatory surface markers CD16 and DNAM-1 on NK cells was prevented by ascites-derived immunoglobulins during NK cell activation. Conclusion: Our data show that high protein concentrations in biological fluids are able to suppress antitumoral activity of NK cells independent from the mechanism mediated by imbalanced electrolytes. The competitive interference between immunoglobulins of ascites and specific therapeutic antibodies could diminish the efficacy of antibody-based therapies and should be considered in antibody-based immunotherapies.

Benchmarking glycoform-resolved affinity separation - mass spectrometry assays for studying FcγRIIIa binding.

The antibody- FcγRIIIa interaction triggers key immunological responses such as antibody dependent cellular cytotoxicity (ADCC), making it highly important for therapeutic mAbs. Due to the direct glycan-glycan interaction with FcγRIIIa receptor, differences in antibody glycosylation can drastically influence the binding affinity. Understanding the differential binding of mAb glycoforms is a very important, yet challenging task due to the co-existence of multiple glycoforms in a sample. Affinity liquid chromatography (AC) and affinity capillary electrophoresis (ACE) hyphenated with mass spectrometry (MS) can provide glycoform-resolved affinity profiles of proteins based on their differences in either dissociation (AC) or equilibrium (ACE) constants. To cross-validate the affinity ranking provided by these complementary novel approaches, both techniques were benchmarked using the same FcγRIIIa constructs. Both approaches were able to assess the mAb - FcγRIIIa interaction in a glycoform selective manner and showed a clear increase in binding for fully versus hemi-fucosylated mAbs. Also, other features, such as increasing affinity with elevated galactosylation or the binding affinity for high mannose glycoforms were consistent. We further applied these approaches to assess the binding towards the F158 allotype of FcγRIIIa, which was not reported before. The FcγRIIIa F158 allotype showed a very similar profile compared to the V158 receptor with the strongest increase in binding due to afucosylation and only a slight increase in binding with additional galactosylation. Both techniques showed a decrease of the binding affinity for high mannose glycoforms for FcγRIIIa F158 compared to the V158 variant. Overall, both approaches provided very comparable results in line with orthogonal methods proving the capabilities of separation-based affinity approaches to study FcγR binding of antibody glycoforms.

Regulatory mechanism of FCGR2A in macrophage polarization and its effects on intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) poses a significant health burden, necessitating a deeper understanding of its molecular underpinnings. Transcriptomic analysis reveals 485 differentially expressed genes (DEGs) associated with IDD, underscoring the importance of immune regulation. Weighted gene co-expression network analysis (WGCNA) identifies a yellow module strongly correlated with IDD, intersecting with 197 DEGs. Protein-protein interaction (PPI) analysis identifies ITGAX, MMP9 and FCGR2A as hub genes, predominantly expressed in macrophages. Functional validation through in vitro and in vivo experiments demonstrates the pivotal role of FCGR2A in macrophage polarization and IDD progression. Mechanistically, FCGR2A knockdown suppresses M1 macrophage polarization and NF-κB phosphorylation while enhancing M2 polarization and STAT3 activation, leading to ameliorated IDD in animal models. This study sheds light on the regulatory function of FCGR2A in macrophage polarization, offering novel insights for IDD intervention strategies. KEY POINTS: This study unveils the role of FCGR2A in intervertebral disc (IVD) degeneration (IDD). FCGR2A knockdown mitigates IDD in cellular and animal models. Single-cell RNA-sequencing uncovers diverse macrophage subpopulations in degenerated IVDs. This study reveals the molecular mechanism of FCGR2A in regulating macrophage polarization. This study confirms the role of the NF-κB/STAT3 pathway in regulating macrophage polarization in IDD.

Therapeutic strategies in FcγIIA receptor-dependent thrombosis and thromboinflammation as seen in heparin-induced thrombocytopenia (HIT) and vaccine-induced immune thrombocytopenia and thrombosis (VITT).

INTRODUCTION: Fcγ-receptors (FcγR) are membrane receptors expressed on a variety of immune cells, specialized in recognition of the Fc part of immunoglobulin G (IgG) antibodies. FcγRIIA-dependent platelet activation in platelet factor 4 (PF4) antibody-related disorders have gained major attention, when these antibodies were identified as the cause of the adverse vaccination event termed vaccine-induced immune thrombocytopenia and thrombosis (VITT) during the COVID-19 vaccination campaign. With the recognition of anti-PF4 antibodies as cause for severe spontaneous and sometimes recurrent thromboses independent of vaccination, their clinical relevance extended far beyond heparin-induced thrombocytopenia (HIT) and VITT. AREAS COVERED: Patients developing these disorders show life-threatening thromboses, and the outcome is highly dependent on effective treatment. This narrative literature review summarizes treatment options for HIT and VITT that are currently available for clinical application and provides the perspective toward new developments. EXPERT OPINION: Nearly all these novel approaches are based on in vitro, preclinical observations, or case reports with only limited implementation in clinical practice. The therapeutic potential of these approaches still needs to be proven in larger cohort studies to ensure treatment efficacy and long-term patient safety.

Essential role of local antibody distribution in mediating bone-resorbing effects.

The link between antibodies and bone mass is debated. Activated IgG, which interacts directly with Fc gamma receptors, stimulates osteoclastogenesis in vitro, and local injection in immune-activated mice leads to bone loss. Multiple myeloma patients with high serum IgG levels have induced osteoclast activation and display bone loss. In addition, bone loss has been linked to serum autoantibodies in autoimmune diseases, including anti-citrullinated protein antibodies (ACPA) in individuals with rheumatoid arthritis (RA). Whether serum IgG or autoantibodies regulate bone mass under healthy conditions is poorly studied. In elderly men, neither serum levels of polyclonal IgG nor autoantibody were associated with areal bone mineral density in the MrOS Sweden study. Repetitive systemic injections of high-dose polyclonal IgG complexes in mice did not exert any discernible impact on bone mineral density. However, repetitive local intra-articular injection of the same IgG complexes led to a localized reduction of trabecular bone density. These results indicate antibodies may only impact bone density when close to the bone, such as within the synovial joint.

A low-temperature SPR-based assay for monoclonal antibody galactosylation and fucosylation assessment using FcγRIIA/B.

Monoclonal antibodies (MAbs) are powerful therapeutic tools in modern medicine and represent a rapidly expanding multibillion USD market. While bioprocesses are generally well understood and optimized for MAbs, online quality control remains challenging. Notably, N-glycosylation is a critical quality attribute of MAbs as it affects binding to Fcγ receptors (FcγRs), impacting the efficacy and safety of MAbs. Traditional N-glycosylation characterization methods are ill-suited for online monitoring of a bioreactor; in contrast, surface plasmon resonance (SPR) represents a promising avenue, as SPR biosensors can record MAb-FcγR interactions in real-time and without labeling. In this study, we produced five lots of differentially glycosylated Trastuzumab (TZM) and finely characterized their glycosylation profile by HILIC-UPLC chromatography. We then compared the interaction kinetics of these MAb lots with four FcγRs including FcγRIIA and FcγRIIB at 5°C and 25°C. When interacting with FcγRIIA/B at low temperature, the differentially glycosylated MAb lots exhibited distinct kinetic behaviors, contrary to room-temperature experiments. Galactosylated TZM (1) and core fucosylated TZM (2) could be discriminated and even quantified using an analytical technique based on the area under the curve of the signal recorded during the dissociation phase of a SPR sensorgram describing the interaction with FcγRIIA (1) or FcγRII2B (2). Because of the rapidity of the proposed method (<5 min per measurement) and the small sample concentration it requires (as low as 30 nM, exact concentration not required), it could be a valuable process analytical technology for MAb glycosylation monitoring.

Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs.

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.

Human Fc gamma receptor IIIA blockade inhibits platelet destruction in a humanized murine model of ITP.

ABSTRACT: Fc gamma receptor (FcγR) IIIA is an important receptor for immunoglobulin G (IgG) and is involved in immune defense mechanisms as well as tissue destruction in some autoimmune diseases including immune thrombocytopenia (ITP). FcγRIIIA on macrophages can trigger phagocytosis of IgG-sensitized platelets, and prior pilot studies observed blockade of FcγRIIIA increased platelet counts in patients with ITP. Unfortunately, although blockade of FcγRIIIA in patients with ITP increased platelet counts, its engagement by the blocking antibody drove serious adverse inflammatory reactions. These adverse events were postulated to originate from the antibody's Fc and/or bivalent nature. The blockade of human FcγRIIIA in vivo with a monovalent construct lacking an active Fc region has not yet been achieved. To effectively block FcγRIIIA in vivo, we developed a high affinity monovalent single-chain variable fragment (scFv) that can bind and block human FcγRIIIA. This scFv (17C02) was expressed in 3 formats: a monovalent fusion protein with albumin, a 1-armed human IgG1 antibody, and a standard bivalent mouse (IgG2a) antibody. Both monovalent formats were effective in preventing phagocytosis of ITP serum-sensitized human platelets. In vivo studies using FcγR-humanized mice demonstrated that both monovalent therapeutics were also able to increase platelet counts. The monovalent albumin fusion protein did not have adverse event activity as assessed by changes in body temperature, whereas the 1-armed antibody induced some changes in body temperature even though the Fc region function was impaired by the Leu234Ala and Leu235Ala mutations. These data demonstrate that monovalent blockade of human FcγRIIIA in vivo can potentially be a therapeutic strategy for patients with ITP.

Point-of-care neutrophil and monocyte surface markers differentiate bacterial from viral infections at the emergency department within 30†min.

Rapid discrimination between viral and bacterial infections in a point-of-care setting will improve clinical outcome. Expression of CD64 on neutrophils (neuCD64) increases during bacterial infections, whereas expression of CD169 on classical monocytes (cmCD169) increases during viral infections. The diagnostic value of automated point-of-care neuCD64 and cmCD169 analysis was assessed for detecting bacterial and viral infections at the emergency department. Additionally, their value as input for machine learning models was studied. A prospective observational cohort study in patients suspected of infection was performed at an emergency department. A fully automated point-of-care flow cytometer measured neuCD64, cmCD169, and additional leukocyte surface markers. Flow cytometry data were gated using the FlowSOM algorithm. Bacterial and viral infections were assessed in standardized clinical care. The sole and combined diagnostic value of the markers was investigated. Clustering based on unsupervised machine learning identified unique patient clusters. Eighty-six patients were included. Thirty-five had a bacterial infection, 30 had a viral infection, and 21 had no infection. neuCD64 was increased in bacterial infections (P < 0.001), with an area under the receiver operating characteristic curve (AUROC) of 0.73. cmCD169 was higher in virally infected patients (P < 0.001; AUROC 0.79). Multivariate analyses incorporating additional markers increased the AUROC for bacterial and viral infections to 0.86 and 0.93, respectively. The additional clustering identified 4 distinctive patient clusters based on infection type and outcome. Automated neuCD64 and cmCD169 determination can discriminate between bacterial and viral infections. These markers can be determined within 30 min, allowing fast infection diagnostics in the acute clinical setting.

Effect of non-surgical periodontal therapy on CD14 + CD16+ monocyte counts in peripheral blood samples: a clinical interventional study.

Monocytes and their macrophage progeny are thought to be involved in tissue and alveolar bone destruction in periodontal disease. It has been documented that the proportion of (CD14 + CD16+) non-classical monocytes in the blood are elevated in chronic periodontitis;A total of 20 chronic generalized periodontitis patients who were otherwise healthy, were recruited for this study. At baseline and 3 weeks after non-surgical periodontal treatment, peripheral blood was obtained to assess the levels of C-reactive protein (CRP) and the proportion of monocyte subsets. Monocyte subsets were assessed using flow cytometry;The mean percentage of CD14 + CD16+ non-classical monocytes in the peripheral blood sample at baseline was 13.95 + 2.09, that reduced to 8.94 + 1.23 3 weeks after non-surgical treatment. A distinct significant reduction in the percentage of non-classical monocytes and a concomitant increase in classical monocytes were observed following periodontal treatment compared to baseline. There was a significant reduction in the all the periodontal parameters and CRP levels 3 weeks post non-surgical periodontal treatment. A positive correlation between CRP and percentage of non-classical monocytes was also observed; Periodontal treatment potentially modulates the host response effectively.

Generic design principles for antibody-based tumour necrosis factor (TNF) receptor 2 (TNFR2) agonists with FcγR-independent agonism.

Background: Selective TNFR2 activation can be used to treat immune pathologies by activating and expanding regulatory T-cells (Tregs) but may also restore anti-tumour immunity by co-stimulating CD8+ T-cells. Oligomerized TNFR2-specific TNF mutants or anti-TNFR2 antibodies can activate TNFR2 but suffer either from poor production and pharmacokinetics or in the case of anti-TNFR2 antibodies typically from the need of FcγR binding to elicit maximal agonistic activity. Methods: To identify the major factor(s) determining FcγR-independent agonism of anti-TNFR2 antibodies, we systematically investigated a comprehensive panel of anti-TNFR2 antibodies and antibody-based constructs differing in the characteristics of their TNFR2 binding domains but also in the number and positioning of the latter. Results: We identified the domain architecture of the constructs as the pivotal factor enabling FcγR-independent, thus intrinsic TNFR2-agonism. Anti-TNFR2 antibody formats with either TNFR2 binding sites on opposing sites of the antibody scaffold or six or more TNFR2 binding sites in similar orientation regularly showed strong FcγR-independent agonism. The affinity of the TNFR2 binding domain and the epitope recognized in TNFR2, however, were found to be of only secondary importance for agonistic activity. Conclusion: Generic design principles enable the generation of highly active bona fide TNFR2 agonists from nearly any TNFR2-specific antibody.

A secretory protein neudesin regulates splenic red pulp macrophages in erythrophagocytosis and iron recycling.

Neudesin, originally identified as a neurotrophic factor, has primarily been studied for its neural functions despite its widespread expression. Using 8-week-old neudesin knockout mice, we elucidated the role of neudesin in the spleen. The absence of neudesin caused mild splenomegaly, shortened lifespan of circulating erythrocytes, and abnormal recovery from phenylhydrazine-induced acute anemia. Blood cross-transfusion and splenectomy experiments revealed that the shortened lifespan of erythrocytes was attributable to splenic impairment. Further analysis revealed increased erythrophagocytosis and decreased iron stores in the splenic red pulp, which was linked to the upregulation of Fcγ receptors and iron-recycling genes in neudesin-deficient macrophages. In vitro analysis confirmed that neudesin suppressed erythrophagocytosis and expression of Fcγ receptors through ERK1/2 activation in heme-stimulated macrophages. Finally, we observed that 24-week-old neudesin knockout mice exhibited severe symptoms of anemia. Collectively, our results suggest that neudesin regulates the function of red pulp macrophages and contributes to erythrocyte and iron homeostasis.

VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages.

Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Vesicle-associated membrane protein 5 (VAMP5), also a plasmalemma SNARE, interacts with SNAP23; however, its precise function in phagocytosis in macrophages remains elusive. To elucidate this aspect, we investigated the characteristics of macrophages in the presence of VAMP5 overexpression or knockdown and found that VAMP5 participates in Fcγ receptor-mediated phagosome formation, although not directly in phagosome maturation. Overexpressed VAMP5 was localized to the early phagosomal membrane but no longer localized to the lysosomal-associated membrane protein 1-positive maturing phagosomal membrane. Analyses using compound-based selective inhibitors demonstrated that VAMP5 dissociation from early phagosomes occurs in a clathrin- and dynamin-dependent manner and is indispensable for SNAP23 function in subsequent membrane fusion during phagosome maturation. Accordingly, to the best of our knowledge, we demonstrate, for the first time, that VAMP5 exerts an immunologically critical function during phagosome formation and maturation via SNARE-based membrane trafficking in macrophages.

Evaluation of HLA-DR expression in monocytes and CD64 in neutrophils as A predictor of SEPSIS/sirs in the infectious-inflammatory process.

Sepsis is a highly fatal disease that affects millions of people worldwide every year. Currently, the diagnosis of sepsis is made by identifying at least two symptoms of systemic inflammatory response syndrome (SIRS), along with confirming the presence of microorganisms using a blood culture examination. Some biomarkers are already used to aid in the diagnosis, such as increased levels of C-reactive protein (CRP), leukocytes, immature granulocytes (IG), and bands. In addition, studies have shown a relationship between the expression of certain antigen receptors in the body's defense cells and its infectious state. CD64 is a receptor expressed in monocytes, and, in cases of infection, its expression is strongly observed in neutrophils. On the other hand, the class II MHC (major histocompatibility complex) marker, HLA-DR (human leukocyte antigen-DR), decreases its expression in monocytes in response to infection. This cohort study was conducted with 77 adult patients from a university hospital, divided into two groups: Non-Sepsis/SIRS and Sepsis/SIRS. The selected samples were analyzed by flow cytometry, identifying the expression of CD64 and HLA-DR according to their MFI, and calculating the sepsis index (SI) for each patient. All three parameters exhibited significant differences in expression between the two groups. When compared to the laboratory tests already in use, the utilization of HLA-DR, CD64, and the new index has shown greater sensitivity and specificity in identifying sepsis. This study contributes to knowledge about the relationship between the expression of antigens on defense cells and sepsis. The use of these biomarkers can help to improve the diagnosis and treatment of sepsis, which may contribute to the reduction of mortality related to the disease.

Fcγ receptors and immunomodulatory antibodies in cancer.

The discovery of both cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) as negative regulators of antitumour immunity led to the development of numerous immunomodulatory antibodies as cancer treatments. Preclinical studies have demonstrated that the efficacy of immunoglobulin G (IgG)-based therapies depends not only on their ability to block or engage their targets but also on the antibody's constant region (Fc) and its interactions with Fcγ receptors (FcγRs). Fc-FcγR interactions are essential for the activity of tumour-targeting antibodies, such as rituximab, trastuzumab and cetuximab, where the killing of tumour cells occurs at least in part due to these mechanisms. However, our understanding of these interactions in the context of immunomodulatory antibodies designed to boost antitumour immunity remains less explored. In this Review, we discuss our current understanding of the contribution of FcγRs to the in vivo activity of immunomodulatory antibodies and the challenges of translating results from preclinical models into the clinic. In addition, we review the impact of genetic variability of human FcγRs on the activity of therapeutic antibodies and how antibody engineering is being utilized to develop the next generation of cancer immunotherapies.

NK cells and the profile of inflammatory cytokines in the peripheral blood of patients with advanced carcinomas.

BACKGROUND: Natural killer (NK) cells are one of the most crucial immune cells that mediate the antitumoral response due to their ability to immediately recognize and eliminate transformed cells. Because of their great cytotoxic activity, the function of NK cells must be robustly regulated to avoid tissue damage. Such regulation is mediated by a coordinated engagement of activating (NKp46) and inhibitory (CD158b) receptors, which tumor cells may use to escape from immunosurveillance. Also, NK cells are generally divided based on surface molecules, such as CD16 and CD56, and can be classified as CD56brightCD16- (regulatory) and CD56dimCD16+ (cytotoxic) NK cells. Here, we aimed to evaluate the frequency and phenotype of circulating NK cells in patients with advanced carcinomas, as well as their systemic cytokine/chemokine and growth factors production. METHODS: Peripheral blood was collected from 24 patients with advanced solid cancer during or after treatment and from 10 healthy donors. The frequency and the expression of activating (NKp46) and inhibitory (CD158b) molecules of CD56brightCD16- and CD56dimCD16+ NK cells were assessed by flow cytometry and the multiplex Luminex platform was used to quantify the secreted factors in peripheral blood serum. RESULTS: Cancer patients had a lower frequency of the cytotoxic CD56dim CD16+ NK cells subset in comparison with healthy controls. Also, the regulatory CD56bright CD16- NKs isolated from cancer patients exhibited a significantly lower expression of NKp46. Among 29 immunological and growth factors analyzed in the peripheral blood of oncologic patients, MCP-1, IP-10, and eotaxin, and VEGF they have presented a higher proportion. The Pearson correlation test showed that IL-12p40 positively correlates with CD56brightCD16- NK cells. We also observed a positive correlation between MCP-1 and the activating marker NKp46, as well as a negative correlation between IP-10 and TNF-α and NKp46. CD158b expression in CD56dimCD16+ was positively correlated with EGF and negatively correlated with MIP-1ß. CONCLUSIONS: Taken together, these results suggest that cancer patients present a shift towards a poorly cytotoxic and less activated NK profile which may contribute to tumor development and progression. The understanding of NK cell biology and soluble factors during tumor development could aid in the design of possible targeting therapeutic approaches.

Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy.

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.