The Contribution of Liver Sinusoidal Endothelial Cells to Clearance of Therapeutic Antibody

Many chronic inflammatory diseases are treated by administration of “biological” therapies in terms of fully human and humanized monoclonal antibodies or Fc fusion proteins. These tools have widespread efficacy and are favored because they generally exhibit high specificity for target with a low toxicity. However, the design of clinically applicable humanized antibodies is complicated by the need to circumvent normal antibody clearance mechanisms to maintain therapeutic dosing, whilst avoiding development of off target antibody dependent cellular toxicity. Classically, professional phagocytic immune cells are responsible for scavenging and clearance of antibody via interactions with the Fc portion. Immune cells such as macrophages, monocytes, and neutrophils express Fc receptor subsets, such as the FcγR that can then clear immune complexes. Another, the neonatal Fc receptor (FcRn) is key to clearance of IgG in vivo and serum half-life of antibody is explicitly linked to function of this receptor. The liver is a site of significant expression of FcRn and indeed several hepatic cell populations including Kupffer cells and liver sinusoidal endothelial cells (LSEC), play key roles in antibody clearance. This combined with the fact that the liver is a highly perfused organ with a relatively permissive microcirculation means that hepatic binding of antibody has a significant effect on pharmacokinetics of clearance. Liver disease can alter systemic distribution or pharmacokinetics of antibody-based therapies and impact on clinical effectiveness, however, few studies document the changes in key membrane receptors involved in antibody clearance across the spectrum of liver disease. Similarly, the individual contribution of LSEC scavenger receptors to antibody clearance in a healthy or chronically diseased organ is not well characterized. This is an important omission since pharmacokinetic studies of antibody distribution are often based on studies in healthy individuals and thus may not reflect the picture in an aging or chronically diseased population. Therefore, in this review we consider the expression and function of key antibody-binding receptors on LSEC, and the features of therapeutic antibodies which may accentuate clearance by the liver. We then discuss the implications of this for the design and utility of monoclonal antibody-based therapies.

Neonatal Fc Receptor–Targeted Therapies in Neurology

Autoantibodies are increasingly recognized for their pathogenic potential in a growing number of neurological diseases. While myasthenia gravis represents the prototypic antibody (Ab)-mediated neurological disease, many more disorders characterized by Abs targeting neuronal or glial antigens have been identified over the past two decades. Depletion of humoral immune components including immunoglobulin G (IgG) through plasma exchange or immunoadsorption is a successful therapeutic strategy in most of these disease conditions. The neonatal Fc receptor (FcRn), primarily expressed by endothelial and myeloid cells, facilitates IgG recycling and extends the half-life of IgG molecules. FcRn blockade prevents binding of endogenous IgG to FcRn, which forces these antibodies into lysosomal degradation, leading to IgG depletion. Enhancing the degradation of endogenous IgG by FcRn-targeted therapies proved to be a powerful therapeutic approach in patients with generalized MG and is currently being tested in clinical trials for several other neurological diseases including autoimmune encephalopathies, neuromyelitis optica spectrum disorders, and inflammatory neuropathies. This review illustrates mechanisms of FcRn-targeted therapies and appraises their potential to treat neurological diseases.

Preserved recognition of Omicron Spike following COVID-19 mRNA vaccination in pregnancy

Summary Background SARS-CoV-2 infection is associated with enhanced disease severity in pregnant women. Despite the potential of COVID-19 vaccines to reduce severe disease, vaccine uptake remained relatively low among pregnant women. Just as coordinated messaging from the CDC and leading obstetrics organizations began to increase vaccine confidence in this vulnerable group, the evolution of SARS-CoV-2 variants of concerns (VOC) including the Omicron VOC raised new concerns about vaccine efficacy, given their ability to escape vaccine-induced neutralizing antibodies. Early data point to a milder disease course following omicron VOC infection in vaccinated individuals. Thus, these data suggest that alternate vaccine induced immunity, beyond neutralization, may continue to attenuate omicron disease, such as antibody-Fc-mediated activity. However, whether vaccine induced antibodies raised in pregnancy continue to bind and leverage Fc-receptors remains unclear. Methods VOC including Omicron receptor binding domain (RBD) or full Spike specific antibody isotype binding titers and FcγR binding were analyzed in pregnant women after the full dose regimen of either Pfizer/BioNtech BNT62b2 (n=10) or Moderna mRNA-1273 (n=10) vaccination using a multiplexing Luminex assay. Findings Comparable, albeit reduced, isotype recognition was observed to the Omicron Spike and receptor binding domain (RBD) following both vaccines. Yet, despite the near complete loss of Fc-receptor binding to the Omicron RBD, Fc-receptor binding was largely preserved to the Omicron Spike. Interpretation Reduced binding titer to the Omicron RBD aligns with observed loss of neutralizing activity. Despite the loss of neutralization, preserved Omicron Spike recognition and Fc-receptor binding potentially continues to attenuate disease severity in pregnant women. Funding NIH and the Bill and Melinda Gates Foundation

Spontaneous Platelet Aggregation in Blood Is Mediated by FcγRIIA Stimulation of Bruton’s Tyrosine Kinase

High platelet reactivity leading to spontaneous platelet aggregation (SPA) is a hallmark of cardiovascular diseases; however, the mechanism underlying SPA remains obscure. Platelet aggregation in stirred hirudin-anticoagulated blood was measured by multiple electrode aggregometry (MEA) for 10 min. SPA started after a delay of 2–3 min. In our cohort of healthy blood donors (n = 118), nine donors (8%) with high SPA (>250 AU*min) were detected. Pre-incubation of blood with two different antibodies against the platelet Fc-receptor (anti-FcγRIIA, CD32a) significantly reduced high SPA by 86%. High but not normal SPA was dose-dependently and significantly reduced by blocking Fc of human IgG with a specific antibody. SPA was completely abrogated by blood pre-incubation with the reversible Btk-inhibitor (BTKi) fenebrutinib (50 nM), and 3 h after intake of the irreversible BTKi ibrutinib (280 mg) by healthy volunteers. Increased SPA was associated with higher platelet GPVI reactivity. Anti-platelet factor 4 (PF4)/polyanion IgG complexes were excluded as activators of the platelet Fc-receptor. Our results indicate that high SPA in blood is due to platelet FcγRIIA stimulation by unidentified IgG complexes and mediated by Btk activation. The relevance of our findings for SPA as possible risk factor of cardiovascular diseases and pathogenic factor contributing to certain autoimmune diseases is discussed.

Regulatory Role of Fc Receptor in mIgM+ B Lymphocyte Phagocytosis in Flounder (Paralichthys olivaceus)

Fc receptor (FcR) is an important opsonin receptor on the surface of immune cells, playing an important role in antibody-dependent cell-mediated immunity. Our previous work found that the FcR of flounder showed a marked expression response in phagocytizing IgM+ B cell, which suggested that FcR might participate in regulating Ig-opsonized phagocytosis. In this paper, in order to elucidate the potential role of FcR in mediating phagocytosis of IgM+ B cell, flounder anti-E. tarda serum was prepared and complement-inactivated for the use of E. tarda opsonization, and the sera of healthy flounder were used as control. Flow cytometric analysis showed that the phagocytosis rates of antiserum-opsonized E. tarda in peripheral blood mIgM+ B lymphocytes were significantly higher than the control group, and higher phagocytosis rates of mIgM+ B lymphocyte could be detected with an increasing incubation time ranging from 1 to 5 h. The phagocytosis rates of antiserum-opsonized E. tarda by mIgM+ B lymphocyte for an incubation time of 1, 3 or 5 h were 51.1, 63.0, and 77.5% respectively, which were significantly higher than the phagocytosis rates in the control groups with 40.2, 50.9, and 63.8%, respectively. While the Fc fragment of IgM on the surface of opsonized E. tarda was blocked by rabbit anti-flounder IgM polyclonal antibodies, phagocytosis rates of mIgM+ B lymphocyte decreased significantly compared with the unblocked group. Moreover, the proportion of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels rose to 32.1% from the control level of 23.0% after phagocytosis of antiserum-opsonized E. tarda. FcγRII and Syk were found to be significantly upregulated, while FcγRIII was significantly downregulated in the mIgM+ B lymphocytes post phagocytosis. Furthermore, when FcγRII of mIgM+ B lymphocytes was blocked by the prepared antibodies, their phagocytosis rate of antiserum-opsonized E. tarda was 39.0%, which was significantly lower than the unblocked group of 54.0%. These results demonstrate that FcR plays a critical role in mediating phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which would facilitate an improved understanding of the regulatory roles of FcR in phagocytosis of teleost B lymphocytes.

Antigenic Modulation of CD6 By Itolizumab Is a New Mechanism for Effector T Cell Inhibition

Introduction Acute Graft-Versus-Host Disease (aGVHD) is a serious complication of hematopoietic stem cell transplantation (HSCT) that is primarily driven by alloreactive T cells. CD6 is a costimulatory receptor primarily expressed on T cells that promotes synapse formation, T cell activation, and migration into tissues by engaging with its ligand, activated leukocyte cell adhesion molecule (ALCAM). CD6 is expressed on reconstituting T cells soon after HSCT (Rambaldi et al., 2019) and early studies have shown that ex-vivo depletion of CD6 + donor cells prior to HSCT decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998). The reduced levels of aGVHD were attributed to an increased prevalence of CD6 - T cells that were less alloreactive (Rasmussen et al., 1994). Therefore, modulating activity of the CD6-ALCAM pathway may ameliorate aGVHD. Itolizumab is a humanized anti-CD6 monoclonal antibody that was previously described to block the engagement of ALCAM, thereby inhibiting T cell activity and trafficking. Here, we elucidate a second and highly novel mechanism in which antibody-mediated loss of CD6 from the surface of T cells results in CD6 low T cells that are hyporesponsive to T cell stimulation. Methods To assess the molecular mechanisms for itolizumab-induced loss of cell surface CD6, peripheral blood mononuclear cells (PBMCs) were exposed to different conditions including treatment with selected protease inhibitors, a membrane inhibitor, or Fc receptor blocking antibodies in the presence of itolizumab. Furthermore, B cells, NK cells, and monocytes were isolated from PBMCs and mixed with T cells in combination with itolizumab to evaluate cell contact requirements for loss. Following treatment, cell surface levels of CD6 protein were assessed by flow cytometry using a non-competing anti-CD6 detection antibody while analysis of full length CD6 was detected by western blot from total protein lysate. The soluble form of CD6 (sCD6) was analyzed by electrochemiluminescence and immunoprecipitation from supernatant of treated cells. Results Following treatment with itolizumab, the levels of cell surface CD6 was reduced as assessed by flow cytometry and western blot. Concurrent with a loss of cell surface CD6, levels of sCD6 in the cell supernatant increased, suggesting CD6 is primarily cleaved from the cell surface rather than internalized. Characterization of the cleaved sCD6 product by western blot revealed a 30KDa product. Itolizumab-induced changes in both levels of cell surface CD6 and sCD6 was abrogated by both 4-benzenesulfonyl fluoride hydrochloride (AEBSF), a serine protease inhibitor and cytochalasin D, an inhibitor of actin polymerization that prevents movement within the cell membrane and blocks endocytic trafficking. This suggests that a membrane-bound serine protease is responsible for cleavage. Itolizumab-induced CD6 cleavage did not occur with T cells alone and required the presence of other immune cells including monocytes and NK cells, but not B cells (Figure 1). In addition, when monocytes and T cells were separated by a membrane, cleavage of CD6 was not observed in the presence of itolizumab, indicating that cell-to-cell contact is required. Furthermore, blocking antibodies indicated that functional Fc receptors, especially FcγRIA, were required, suggesting that binding of itolizumab to both an Fc receptor on monocytes and CD6 on T cells predicated the cleavage event. The resulting CD6 low T cells were hyporesponsive to T cell stimulation, even in the absence of itolizumab, as indicated by reduced expression of PD-1, CD71 and CD25 as well as inflammatory cytokines (Figure 2). Inhibition was observed for both naïve and effector/memory T cell subsets. Conclusions Our results reveal a novel mechanism of antigenic modulation by itolizumab in which CD6 is cleaved from the T cell surface and released in a soluble form. Cleavage of CD6 occurs via a membrane-bound serine protease and appears dependent upon the engagement of itolizumab with Fc receptor(s) present on monocytes and NK cells. The loss of cell surface CD6 results in T cells with reduced responses to TCR-mediated stimulation. As such, treatment of aGVHD patients with itolizumab may reduce the alloreactivity of donor T cells, ameliorating disease symptoms and improving the clinical outcomes in these patients. Figure 1 Figure 1. Disclosures Chu: Equillium: Current Employment. Marrocco: Equillium, Inc.: Current Employment. Tiet: Equillium, Inc.: Current Employment. Ampudia: Equillium, Inc.: Current Employment. Connelly: Equillium: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Ng: Equillium: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months.

Fc-Mediated Complement Activation Is Associated with Macrophage Trafficking and Induction of Neutrophil Extracellular Traps in Transfusion-Related Acute Lung Injury

Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-related fatalities without available therapies. The pulmonary endothelium is damaged in TRALI, through incompletely understood pathogenic mechanisms, resulting in pulmonary edema. Generally, anti-leukocyte antibodies or biological response modifiers in the transfusion product, in combination with predisposing risk factors in the transfused recipient (e.g. inflammation), are responsible for initiation of TRALI. Remarkably, not all anti-leukocyte antibodies cause TRALI. In a previous in vitro study, we identified increased Fc-mediated complement activation to be a key feature of murine TRALI-inducing antibody 34-1-2S (anti-H-2K d) compared to TRALI-resistant antibody SF1.1.10 (anti-H-2K d) (Zeeuw van der Laan et al, Blood Adv 2020). In the current study, we further explored antibody-mediated TRALI mechanisms in vivo using our previously established TRALI mouse model in which mice are pre-depleted of protective CD4+ T cells and primed with LPS, followed by infusion of antibody 34-1-2S (Kapur et al, Blood 2017, Blood Adv 2018, Blood 2019). A key read-out for TRALI was the lung wet/dry weight ratio (Lung W/D, measure for pulmonary edema). We found that in vitro antibody-mediated complement activation was associated with in vivo antibody-mediated TRALI. 34-1-2S caused severe TRALI (complement activation: +++, Lung W/D: 7.4 ± 0.21), while SF1.1.10 did not cause TRALI (complement activation: +, Lung W/D: 4.68 ± 0.16). Furthermore, Fc-deglycosylated 34-1-2S still caused significant TRALI (complement activation: ++, in vivo Lung W/D: 5.16 ± 0.52). TRALI development was fully Fc-dependent as 34-1-2S-Fab did not cause any TRALI (no complement activation, Lung W/D: 4.35 ± 0.18). Importantly, we found significantly increased levels of complement C1q-C4 complexes in plasma samples of TRALI patients (n=46) compared to healthy controls (n=25): 6.49 ± 5.27 vs 3.84 ± 2.27 AU/ml, respectively, P: 0.0005,***. Similarly, C5a levels were significantly elevated in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 2.52 ± 2.17 vs 1.37 ± 1.08 ng/ml, respectively, P: 0.0006,***. To further dissect the effect of the Fc-part of 34-1-2S in TRALI-induction, we aim to investigate the contribution of Fc-mediated complement activation vs Fc-receptor interaction. We therefore successfully generated chimeric variants of 34-1-2S with a humanized IgG1 Fc-domain containing mutations making them functionally complement dead (K322A), Fc-receptor dead (dG236) or both complement and Fc-receptor dead (PG LALA). We are currently investigating the effects of these variants on in vivo TRALI induction. We next investigated how the in vivo murine TRALI reaction was related to numbers of macrophages, monocytes and neutrophils in blood and lungs. We found that 34-1-2S-TRALI was associated with significantly decreased levels of macrophages in the lungs and increased levels in blood, compared to mice infused with PBS, SF1.1.10 or 34-1-2S-Fab, suggesting that Fc-mediated complement activation and TRALI induction is related to macrophage trafficking from lungs to blood. We did not observe any significant differences between blood and lung neutrophil levels of 34-1-2S-TRALI mice compared to SF1.1.10 TRALI-resistant mice. We hypothesized that in TRALI neutrophils undergo formation of neutrophil extracellular traps (NETs) induced by complement. We observed that C5a enabled potent neutrophil-chemotaxis in vitro (P: 0.0048,**). In addition, using direct immunofluorescence staining of extracellular DNA and Citrullinated histone H3, we observed that both LPS and C5a on their own could induce NET formation in vitro, which was synergistically increased with a combination of both LPS and C5a (P: 0.0417,*), as occurring during TRALI. Furthermore, we found increased levels of NETs to be present in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 1.64 ± 0.97 vs 0.80 ± 0.34 MPO-DNA OD, respectively, P: 0.0002,***. Finally, we targeted the C5a-receptor (C5aR) using a C5aR-antagonist in our TRALI mouse model. Surprisingly, this did not prevent but even worsened TRALI (P: 0.0398,*), with elevated levels of blood monocytes and macrophages. This suggests that an approach directly targeting complement components may be a more promising therapeutic strategy to explore in combatting TRALI. Disclosures No relevant conflicts of interest to declare.

Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary macrophages using biophysical fluorescence techniques

The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin and IgG and contributes to their long plasma half-life. We recently identified a FcRn-dependent, recycling pathway from macropinosomes in macrophages (Toh et al, 2019), however, little is known about the dynamics of intracellular FcRn-ligand interactions to promote recycling. Here we demonstrate a multiplexed biophysical fluorescent microscopy approach to resolve the spatiotemporal dynamics of albumin-FcRn interactions in living bone marrow-derived macrophages (BMDMs). We used the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) to detect the interaction of a FcRn-mCherry fusion protein with endocytosed Alexa Fluor 488-labelled human serum albumin (HSA-AF488) in BMDMs, and Raster Image Correlation Spectroscopy (RICS) analysis of single fluorescent-labelled albumin molecules to monitor the diffusion kinetics of internalised albumin. Our data identified a major fraction of immobile HSA-AF488 molecules in endosomal structures of human FcRn-positive mouse macrophages and an increase in FLIM- FRET following endocytosis, including detection of FRET in tubular-like structures. A non-binding mutant of albumin showed minimum FLIM-FRET and high mobility. These data reveal the kinetics of FcRn-ligand binding within endosomal structures for recruitment into transport carriers for recycling. These approaches have wide applicability for analyses of intracellular ligand-receptor interactions.