Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.

MiR-23a Regulates Skin Langerhans Cell Phagocytosis and Inflammation-Induced Langerhans Cell Repopulation.

Langerhans cells (LCs) are skin-resident macrophage that act similarly to dendritic cells for controlling adaptive immunity and immune tolerance in the skin, and they are key players in the development of numerous skin diseases. While TGF-ß and related downstream signaling pathways are known to control numerous aspects of LC biology, little is known about the epigenetic signals that coordinate cell signaling during LC ontogeny, maintenance, and function. Our previous studies in a total miRNA deletion mouse model showed that miRNAs are critically involved in embryonic LC development and postnatal LC homeostasis; however, the specific miRNA(s) that regulate LCs remain unknown. miR-23a is the first member of the miR-23a-27a-24-2 cluster, a direct downstream target of PU.1 and TGF-b, which regulate the determination of myeloid versus lymphoid fates. Therefore, we used a myeloid-specific miR-23a deletion mouse model to explore whether and how miR-23a affects LC ontogeny and function in the skin. We observed the indispensable role of miR-23a in LC antigen uptake and inflammation-induced LC epidermal repopulation; however, embryonic LC development and postnatal homeostasis were not affected by cells lacking miR23a. Our results suggest that miR-23a controls LC phagocytosis by targeting molecules that regulate efferocytosis and endocytosis, whereas miR-23a promotes homeostasis in bone marrow-derived LCs that repopulate the skin after inflammatory insult by targeting Fas and Bcl-2 family proapoptotic molecules. Collectively, the context-dependent regulatory role of miR-23a in LCs represents an extra-epigenetic layer that incorporates TGF-b- and PU.1-mediated regulation during steady-state and inflammation-induced repopulation.

Dysregulated CD38 expression in blood and skin immune cells of patients with hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a multifactorial, inflammatory skin disease. Increased systemic inflammatory comorbidities and serum cytokines highlight systemic inflammation as a feature of HS. However, the specific immune cell subsets contributing to systemic and cutaneous inflammation have not been resolved. Here, we generated whole-blood immunomes by mass cytometry. We performed a meta-analysis of RNA-seq data, immunohistochemistry, and imaging mass cytometry to characterize the immunological landscape of skin lesions and perilesions from patients with HS. Blood from patients with HS exhibited lower frequencies of natural killer cells, dendritic cells, and classical (CD14+CD16-) and nonclassical (CD14-CD16+) monocytes, as well as higher frequencies of Th17 cells and intermediate (CD14+CD16+) monocytes than blood from healthy controls. Classical and intermediate monocytes from patients with HS had increased expression of skin-homing chemokine receptors. Furthermore, we identified a CD38+ intermediate monocyte subpopulation that was more abundant in the immunome of blood from patients with HS. Meta-analysis of RNA-seq data found higher CD38 expression in lesional HS skin than in perilesional skin, and markers of classical monocyte infiltration. Imaging mass cytometry showed that CD38+ classical monocytes and CD38+ monocyte-derived macrophages were more abundant in lesional HS skin. Overall, we report targeting CD38 may be worth pursuing in clinical trials.

Dysregulated CD38 expression in blood and skin immune cells of patients with hidradenitis suppurativa.

Background: Hidradenitis suppurativa (HS) is a multifactorial, inflammatory skin disease. Increased systemic inflammatory comorbidities and serum cytokines highlight systemic inflammation as a feature of HS. However, the specific immune cell subsets contributing to systemic and cutaneous inflammation have not been resolved. Objective: Identify features of peripheral and cutaneous immune dysregulation. Methods: Here, we generated whole-blood immunomes by mass cytometry. We performed a meta-analysis of RNA-seq data, immunohistochemistry, and imaging mass cytometry to characterize the immunological landscape of skin lesions and perilesions from patients with HS. Results: Blood from patients with HS exhibited lower frequencies of natural killer cells, dendritic cells, and classical (CD14+CD16-) and nonclassical (CD14-CD16+) monocytes, as well as higher frequencies of Th17 cells and intermediate (CD14+CD16+) monocytes than blood from healthy controls. Classical and intermediate monocytes from patients with HS had increased expression of skin-homing chemokine receptors. Furthermore, we identified a CD38+ intermediate monocyte subpopulation that was more abundant in the immunome of blood from patients with HS. Meta-analysis of RNA-seq data found higher CD38 expression in lesional HS skin than in perilesional skin, and markers of classical monocyte infiltration. Imaging mass cytometry showed that CD38+ classical monocytes and CD38+ monocyte-derived macrophages were more abundant in lesional HS skin. Conclusion: Overall, we report targeting CD38 may be worth pursuing in clinical trials. Key Messages: 3.Monocyte subsets express markers of activation in circulation and HS lesionsTargeting CD38 may be a viable strategy for treating systemic and cutaneous inflammation in patients with HS. Capsule Summary: 4.Dysregulated immune cells in patients with HS express CD38 and may be targeting by anti-CD38 immunotherapy.

Facile generation of biepitopic antibodies with intrinsic agonism for activating receptors in the tumor necrosis factor superfamily.

Agonist antibodies that activate cellular receptors are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.

Surface translocation of ACE2 and TMPRSS2 upon TLR4/7/8 activation is required for SARS-CoV-2 infection in circulating monocytes.

Infection of human peripheral blood cells by SARS-CoV-2 has been debated because immune cells lack mRNA expression of both angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease type 2 (TMPRSS2). Herein we demonstrate that resting primary monocytes harbor abundant cytoplasmic ACE2 and TMPRSS2 protein and that circulating exosomes contain significant ACE2 protein. Upon ex vivo TLR4/7/8 stimulation, cytoplasmic ACE2 was quickly translocated to the monocyte cell surface independently of ACE2 transcription, while TMPRSS2 surface translocation occurred in conjunction with elevated mRNA expression. The rapid translocation of ACE2 to the monocyte cell surface was blocked by the endosomal trafficking inhibitor endosidin 2, suggesting that endosomal ACE2 could be derived from circulating ACE2-containing exosomes. TLR-stimulated monocytes concurrently expressing ACE2 and TMPRSS2 on the cell surface were efficiently infected by SARS-CoV-2, which was significantly mitigated by remdesivir, TMPRSS2 inhibitor camostat, and anti-ACE2 antibody. Mass cytometry showed that ACE2 surface translocation in peripheral myeloid cells from patients with severe COVID-19 correlated with its hyperactivation and PD-L1 expression. Collectively, TLR4/7/8-induced ACE2 translocation with TMPRSS2 expression makes circulating monocytes permissive to SARS-CoV-2 infection.

Histone deacetylase 3 controls lung alveolar macrophage development and homeostasis.

Alveolar macrophages (AMs) derived from embryonic precursors seed the lung before birth and self-maintain locally throughout adulthood, but are regenerated by bone marrow (BM) under stress conditions. However, the regulation of AM development and maintenance remains poorly understood. Here, we show that histone deacetylase 3 (HDAC3) is a key epigenetic factor required for AM embryonic development, postnatal homeostasis, maturation, and regeneration from BM. Loss of HDAC3 in early embryonic development affects AM development starting at E14.5, while loss of HDAC3 after birth affects AM homeostasis and maturation. Single-cell RNA sequencing analyses reveal four distinct AM sub-clusters and a dysregulated cluster-specific pathway in the HDAC3-deficient AMs. Moreover, HDAC3-deficient AMs exhibit severe mitochondrial oxidative dysfunction and deteriorative cell death. Mechanistically, HDAC3 directly binds to Pparg enhancers, and HDAC3 deficiency impairs Pparg expression and its signaling pathway. Our findings identify HDAC3 as a key epigenetic regulator of lung AM development and homeostasis.

Widespread gene transfer to malignant gliomas with In vitro-to-In vivo correlation.

Gene therapy of malignant gliomas has shown a lack of clinical success to date due in part to inability of conventional gene vectors to achieve widespread gene transfer throughout highly disseminated tumor areas within the brain. Here, we demonstrate that newly engineered polymer-based DNA-loaded nanoparticles (DNA-NP) possessing small particle diameters (~50 nm) and non-adhesive surface polyethylene glycol (PEG) coatings efficiently penetrate brain tumor tissue as well as healthy brain parenchyma. Specifically, this brain-penetrating nanoparticle (BPN), following intracranial administration via convection enhanced delivery (CED), provides widespread transgene expression in heathy rodent striatum and an aggressive brain tumor tissue established orthotopically in rats. The ability of BPN to efficiently traverse both tissues is of great importance as the highly invasive glioma cells infiltrated into normal brain tissue are responsible for tumor recurrence. Of note, the transgene expression within the orthotopic tumor tissue occurred preferentially in glioma cells over microglial cells. We also show that three-dimensional (3D) multicellular spheroids established with malignant glioma cells, unlike conventional two-dimensional (2D) cell cultures, serve as an excellent in vitro model reliably predicting gene vector behaviors in vivo. Briefly, DNA-NP possessing greater surface PEG coverage exhibited more uniform and higher-level transgene expression both in the 3D model and in vivo, whereas the trend was opposite in 2D culture. The finding here alerts that gene transfer studies based primarily on 2D cultures should be interpreted with caution and underscores the relevance of 3D models for screening newly engineered gene vectors prior to their in vivo evaluation.