Gsk3 is a metabolic checkpoint regulator in B cells.

B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.

Generation and Characterization of Mirikizumab, a Humanized Monoclonal Antibody Targeting the p19 Subunit of IL-23.

Interleukin (IL)-23 exists as a heterodimer consisting of p19 and p40 and is a key cytokine for promoting inflammatory responses in a variety of target organs. IL-23 plays a key role in the differentiation and maintenance of T helper 17 cells, and deregulation of IL-23 can result in autoimmune pathologies of the skin, lungs, and gut. This study describes the generation and characterization of mirikizumab (miri), a humanized IgG4 monoclonal antibody directed against the p19 subunit of IL-23. Miri binds human and cynomolgus monkey IL-23 with high affinity and binds rabbit IL-23 weakly but does not bind to rodent IL-23 or the other IL-23 family members IL-12, IL-27, or IL-35. Miri effectively inhibits the interaction of IL-23 with its receptor, and potently blocks IL-23-induced IL-17 production in cell-based assays while preserving the function of IL-12. In both local and systemic in vivo mouse models, miri blocked IL-23-induced keratin mRNA or IL-17 production, respectively. These data provide a comprehensive preclinical characterization of miri, for which efficacy and safety have been demonstrated in human clinical trials for psoriasis, ulcerative colitis, and Crohn's disease. SIGNIFICANCE STATEMENT: This article describes the generation and characterization of mirikizumab, a high affinity, neutralizing IgG4 variant monoclonal antibody that is under development for the treatment of ulcerative colitis and Crohn's disease. Neutralization of interleukin (IL)-23 is achieved by preventing the binding of IL-23 p19 subunit to the IL-23 receptor and does not affect the IL-12 pathway.

Relationship between gene expression patterns from nasopharyngeal swabs and serum biomarkers in patients hospitalized with COVID-19, following treatment with the neutralizing monoclonal antibody bamlanivimab.

BACKGROUND: A thorough understanding of a patient's inflammatory response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is crucial to discerning the associated, underlying immunological processes and to the selection and implementation of treatment strategies. Defining peripheral blood biomarkers relevant to SARS-CoV-2 infection is fundamental to detecting and monitoring this systemic disease. This safety-focused study aims to monitor and characterize the immune response to SARS-CoV-2 infection via analysis of peripheral blood and nasopharyngeal swab samples obtained from patients hospitalized with Coronavirus disease 2019 (COVID-19), in the presence or absence of bamlanivimab treatment. METHODS: 23 patients hospitalized with COVID-19 were randomized to receive a single dose of the neutralizing monoclonal antibody, bamlanivimab (700 mg, 2800 mg or 7000 mg) or placebo, at study initiation (Clinical Trial; NCT04411628). Serum samples and nasopharyngeal swabs were collected at multiple time points over 1 month. A Proximity Extension Array was used to detect inflammatory profiles from protein biomarkers in the serum of hospitalized COVID-19 patients relative to age/sex-matched healthy controls. RNA sequencing was performed on nasopharyngeal swabs. A Luminex serology assay and Elecsys® Anti-SARS-CoV-2 immunoassay were used to detect endogenous antibody formation and to monitor seroconversion in each cohort over time. A mixed model for repeated measures approach was used to analyze changes in serology and serum proteins over time. RESULTS: Levels of IL-6, CXCL10, CXCL11, IFNγ and MCP-3 were > fourfold higher in the serum of patients with COVID-19 versus healthy controls and linked with observations of inflammatory and viral-induced interferon response genes detected in nasopharyngeal swab samples from the same patients. While IgA and IgM titers peaked around 7 days post-dose, IgG titers remained high, even after 28 days. Changes in biomarkers over time were not significantly different between the bamlanivimab and placebo groups. CONCLUSIONS: Similarities observed between nasopharyngeal gene expression patterns and peripheral blood biomarker profiles reveal a connection between the circulation and processes in the nasopharyngeal cavity, reinforcing the potential utility of systemic blood biomarker profiling for therapeutic monitoring of patient response. Serological antibody responses in patients correlated closely with reductions in the COVID-19 inflammatory protein biomarker signature. Bamlanivimab did not affect the biomarker dynamics in this hospitalized patient population.

Characterization of the cytokine storm reflects hyperinflammatory endothelial dysfunction in COVID-19.

BACKGROUND: Physicians treating patients with coronavirus disease 2019 (COVID-19) increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared with healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. OBJECTIVE: To identify and characterize the host inflammatory response to severe acute respiratory syndrome coronavirus 2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease in patients stratified as mild, moderate, and severe versus matched healthy controls. METHODS: Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. RESULTS: Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within patients with COVID-19, and strong biomarker association with patient response as measured by Ordinal Scale. As patients progress, we observe statistically significant dysregulation of IFN-γ, IL-1RA, IL-6, IL-10, IL-19, monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, CXCL9, CXCL10, CXCL5, ENRAGE, and poly (ADP-ribose) polymerase 1. Furthermore, in a limited series of patients who were sampled frequently, confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. CONCLUSIONS: These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of severe acute respiratory syndrome coronavirus 2 pathogenesis and the host response.

Human mast cells release the migraine-inducing factor pituitary adenylate cyclase-activating polypeptide (PACAP).

Background Many patients with migraines suffer from allergies and vice versa, suggesting a relationship between biological mechanisms of allergy and migraine. It was proposed many years ago that mast cells may be involved in the pathophysiology of migraines. We set out to investigate the relationship between mast cell activation and known neurogenic peptides related to migraine. Methods Cultured human mast cells were assayed for the presence of neuropeptides and their receptors at the RNA and protein level. Immunohistochemistry analyses were performed on tissue resident and cultured mast cells. Mast cell degranulation assays were performed and pituitary adenylate cyclase-activating polypeptide (PACAP) activity was measured with a bioassay. Results We found that cultured and tissue resident human mast cells contain PACAP in cytoplasmic granules. No other neurogenic peptide known to be involved in migraine was detected, nor did mast cells express the receptors for PACAP or other neurogenic peptides. Furthermore, mast cell degranulation through classic IgE-mediated allergic mechanisms led to the release of PACAP. The PACAP released from mast cells was biologically active, as demonstrated using PACAP receptor reporter cell lines. We confirmed existing literature that mast cell degranulation can also be induced by several neurogenic peptides, which also resulted in PACAP release. Conclusion Our data provides a potential biological explanation for the association between allergy and migraine by demonstrating the release of biologically active PACAP from mast cells.

IgG-Immune Complexes Promote B Cell Memory by Inducing BAFF.

Memory B cell responses are vital for protection against infections but must also be regulated to prevent autoimmunity. Cognate T cell help, somatic hypermutation, and affinity maturation within germinal centers (GCs) are required for high-affinity memory B cell formation; however, the signals that commit GC B cells to the memory pool remain unclear. In this study, we identify a role for IgG-immune complexes (ICs), FcγRs, and BAFF during the formation of memory B cells in mice. We found that early secretion of IgG in response to immunization with a T-dependent Ag leads to IC-FcγR interactions that induce dendritic cells to secrete BAFF, which acts at or upstream of Bcl-6 in activated B cells. Loss of CD16, hematopoietic cell-derived BAFF, or blocking IC:FcγR regions in vivo diminished the expression of Bcl-6, the frequency of GC and memory B cells, and secondary Ab responses. BAFF also contributed to the maintenance and/or expansion of the follicular helper T cell population, although it was dispensable for their formation. Thus, early Ab responses contribute to the optimal formation of B cell memory through IgG-ICs and BAFF. Our work defines a new role for FcγRs in GC and memory B cell responses.

Elevated levels of Interleukin (IL)-33 induce bone pathology but absence of IL-33 does not negatively impact normal bone homeostasis.

Interleukin (IL)-33 is a member of the IL-1 family. IL-33 effects are mediated through its receptor, ST2 and IL-1RAcP, and its signaling induces the production of a number of pro-inflammatory mediators, including TNFα, IL-1ß, IL-6, and IFN-γ. There are conflicting reports on the role of IL-33 in bone homeostasis, with some demonstrating a bone protective role for IL-33 whilst others show that IL-33 induces inflammatory arthritis with concurrent bone destruction. To better clarify the role IL-33 plays in bone biology in vivo, we studied IL-33 KO mice as well as mice in which the cytokine form of IL-33 was overexpressed. Mid-femur cortical bone mineral density (BMD) and bone strength were similar in the IL-33 KO mice compared to WT animals during the first 8months of life. However, in the absence of IL-33, we observed higher BMD in lumbar vertebrae and distal femur in female mice. In contrast, overexpression of IL-33 resulted in a marked and rapid reduction of bone volume, mineral density and strength. Moreover, this was associated with a robust increase in inflammatory cytokines (including IL-6 and IFN-γ), suggesting the bone pathology could be a direct effect of IL-33 or an indirect effect due to the induction of other mediators. Furthermore, the detrimental bone effects were accompanied by increases in osteoclast number and the bone resorption marker of C-terminal telopeptide collagen-I (CTX-I). Together, these results demonstrate that absence of IL-33 has no negative consequences in normal bone homeostasis while high levels of circulating IL-33 contributes to pathological bone loss.

Translational Pharmacodynamics of Calcitonin Gene-Related Peptide Monoclonal Antibody LY2951742 in a Capsaicin-Induced Dermal Blood Flow Model.

LY2951742, a monoclonal antibody targeting calcitonin gene-related peptide (CGRP), is being developed for migraine prevention and osteoarthritis pain. To support the clinical development of LY2951742, capsaicin-induced dermal blood flow (DBF) was used as a target engagement biomarker to assess CGRP activity in nonhuman primates and healthy volunteers. Inhibition of capsaicin-induced DBF in nonhuman primates, measured with laser Doppler imaging, was dose dependent and sustained for at least 29 days after a single intravenous injection of the CGRP antibody. This information was used to generate a pharmacokinetic/pharmacodynamic model, which correctly predicted inhibition of capsaicin-induced DBF in humans starting at a single subcutaneous 5-mg dose. As expected, the degree of inhibition in capsaicin-induced DBF increased with higher LY2951742 plasma concentrations. Utilization of this pharmacodynamic biomarker with pharmacokinetic data collected in phase I studies provided the dose-response relationship that assisted in dose selection for the phase II clinical development of LY2951742.

Dysregulated NUB1 and Neddylation Enhances Rheumatoid Arthritis Fibroblast-Like Synoviocyte Inflammatory Responses.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) contribute to the pathogenesis of rheumatoid arthritis (RA), in part due to activation of the proinflammatory transcription factor NF-κB. Neddylation is modulated by the negative regulator of ubiquitin-like protein (NUB) 1. We determined whether NUB1 and neddylation are aberrant in the models with RA FLS, thereby contributing to their aggressive phenotype. METHODS: Models with RA or osteoarthritis (OA) FLS were obtained from arthroplasty synovia. Real-time quantitative polymerase chain reaction and Western blot analysis assessed gene and protein expression, respectively. NUB1 was overexpressed using an expression vector. NF-κB activation was assessed by stimulating FLS with interleukin (IL)-1ß. Neddylation inhibitor (MLN4924) and proteasome inhibitor were used in migration and gene expression assays. MLN4924 was used in the model with K/BxN serum-transfer arthritis. RESULTS: Enhanced H3K27ac and H3K27me3 peaks were observed in the NUB1 promoter in the OA FLS compared with the RA FLS. NUB1 was constitutively expressed by FLS, but induction by IL-1ß was significantly greater in the OA FLS. The ratio of neddylated cullin (CUL) 1 to nonneddylated CUL1 was lower in the OA FLS than the RA FLS. NUB1 overexpression decreased NF-κB nuclear translocation and IL-6 messenger RNA (mRNA) in IL-1ß-stimulated the RA FLS. MLN4924 decreased CUL1 neddylation, NF-κB nuclear translocation, and IL-6 mRNA in IL-1ß-stimulated the RA FLS. MLN4924 significantly decreased arthritis severity in the model with K/BxN serum-transfer arthritis. CONCLUSION: CUL1 neddylation and NUB1 induction is dysregulated in the models with RA, which increases FLS activation. Inhibition of neddylation is an effective therapy in an animal model of arthritis. These data suggest that the neddylation system contributes to the pathogenesis of RA and that regulation of neddylation could be a novel therapeutic approach.

Laser Capture Microscopy RNA Sequencing for Topological Mapping of Synovial Pathology During Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. METHODS: Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate. RESULTS: RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.

Binding, Neutralization and Internalization of the Interleukin-13 Antibody, Lebrikizumab.

INTRODUCTION: IL-13 is the primary upregulated cytokine in atopic dermatitis (AD) skin and is the pathogenic mediator driving AD pathophysiology. Lebrikizumab, tralokinumab and cendakimab are therapeutic monoclonal antibodies (mAb) that target IL-13. METHODS: We undertook studies to compare in vitro binding affinities and cell-based functional activities of lebrikizumab, tralokinumab and cendakimab. RESULTS: Lebrikizumab bound IL-13 with higher affinity (as determined using surface plasma resonance) and slower off-rate. It was more potent in neutralizing IL-13-induced effects in STAT6 reporter and primary dermal fibroblast periostin secretion assays than either tralokinumab or cendakimab. Live imaging confocal microscopy was employed to determine the mAb effects on IL-13 internalization into cells via the decoy receptor IL-13Rα2, using A375 and HaCaT cells. The results showed that only the IL-13/lebrikizumab complex was internalized and co-localized with lysosomes, whereas IL-13/tralokinumab or IL-13/cendakimab complexes did not internalize. CONCLUSION: Lebrikizumab is a potent, neutralizing high-affinity antibody with a slow disassociation rate from IL-13. Additionally, lebrikizumab does not interfere with IL-13 clearance. Lebrikizumab has a different mode of action to both tralokinumab and cendakimab, possibly contributing to the clinical efficacy observed by lebrikizumab in Ph2b/3 AD studies.

Evaluation of plasma IL-21 as a potential biomarker for type 1 diabetes progression.

IL-21 is a multifunctional cytokine linked with the pathophysiology of several autoimmune diseases, including type 1 diabetes. In this study, our aim was to examine plasma IL-21 levels in individuals at different stages of type 1 diabetes progression. We measured plasma IL-21 levels, as well as levels of other key pro-inflammatory cytokines (IL-17A, TNF-α and IL-6), from 37 adults with established type 1 diabetes and 46 healthy age-matched adult controls, as well as from 53 children with newly diagnosed type 1 diabetes, 48 at-risk children positive for type 1 diabetes-associated autoantibodies and 123 healthy age-matched pediatric controls using the ultrasensitive Quanterix SiMoA technology. Adults with established type 1 diabetes had higher plasma IL-21 levels compared to healthy controls. However, the plasma IL-21 levels showed no statistically significant correlation with clinical variables, such as BMI, C-peptide, HbA1c, or hsCRP levels, evaluated in parallel. In children, plasma IL-21 levels were almost ten times higher than in adults. However, no significant differences in plasma IL-21 levels were detected between healthy children, autoantibody-positive at-risk children, and children with newly diagnosed type 1 diabetes. In conclusion, plasma IL-21 levels in adults with established type 1 diabetes were increased, which may be associated with autoimmunity. The physiologically high plasma IL-21 levels in children may, however, reduce the potential of IL-21 as a biomarker for autoimmunity in pediatric subjects.

Proteomic characterisations of ulcerative colitis endoscopic biopsies associate with clinically relevant histological measurements of disease severity.

AIMS AND METHODS: Accurate protein measurements using formalin-fixed biopsies are needed to improve disease characterisation. This feasibility study used targeted and global mass spectrometry (MS) to interrogate a spectrum of disease severities using 19 ulcerative colitis (UC) biopsies. RESULTS: Targeted assays for CD8, CD19, CD132 (interleukin-2 receptor subunit gamma/common cytokine receptor gamma chain), FOXP3 (forkhead box P3) and IL17RA (interleukin 17 receptor A) were successful; however, assays for IL17A (interleukin 17A), IL23 (p19) (interleukin 23, alpha subunit p19) and IL23R (interleukin 23 receptor) did not permit target detection. Global proteome analysis (4200 total proteins) was performed to identify pathways associated with UC progression. Positive correlation was observed between histological scores indicating active colitis and neutrophil-related measurements (R2=0.42-0.72); inverse relationships were detected with cell junction targets (R2=0.49-0.71) and ß-catenin (R2=0.51-0.55) attributed to crypt disruption. An exploratory accuracy assessment with Geboes Score and Robarts Histopathology Index cut-offs produced sensitivities/specificities of 72.7%/75.0% and 100.0%/81.8%, respectively. CONCLUSIONS: Pathologist-guided MS assessments provide a complementary approach to histological scoring systems. Additional studies are indicated to verify the utility of this novel approach.

COVID-19 symptom relationship to antibody response and ACE2 neutralization in recovered health systems employees before and after mRNA BNT162b2 COVID-19 vaccine.

BACKGROUND: The humoral response to SARS-CoV-2 can provide immunity and prevent reinfection. However, less is known about how the diversity, magnitude, and length of the antibody response after a primary infection is associated with symptoms, post-infection immunity, and post-vaccinated immunity. METHODS: Cook County Health employees provided blood samples and completed an online survey 8-10 weeks after a PCR-confirmed positive SARS-CoV-2 test (pre-vaccinated, N = 41) and again, 1-4 weeks after completion of a 2-dose series mRNA BNT162b2 COVID-19 vaccine (post-vaccinated, N = 27). Associations were evaluated between SARS-CoV-2 antibody titers, participant demographics, and clinical characteristics. Antibody titers and angiotensin-converting enzyme 2 (ACE2) neutralization were compared before and after the mRNA BNT162b2 COVID-19 vaccine. RESULTS: Antibody titers to the spike protein (ST4), receptor binding domain (RBD), and RBD mutant D614G were significantly associated with anosmia and ageusia, cough, and fever. Spike protein antibody titers and ACE2 neutralization were significantly higher in participants that presented with these symptoms. Antibody titers to the spike protein N-terminal domain (NTD), RBD, and ST4, and ACE2 IC50 were significantly higher in all post-vaccinated participant samples compared to pre-vaccinated participant sample, and not dependent on previously reported symptoms. CONCLUSIONS: Spike protein antibody titers and ACE2 neutralization are associated with the presentation of anosmia and ageusia, cough, and fever after SARS-CoV-2 infection. Symptom response to previous SARS-CoV-2 infection did not influence the antibody response from subsequent vaccination. These results suggest a relationship between infection severity and the magnitude of the immune response and provide meaningful insights into COVID-19 immunity according to discrete symptom presentation.

The anti-SARS-CoV-2 monoclonal antibody bamlanivimab minimally affects the endogenous immune response to COVID-19 vaccination.

As the coronavirus disease 2019 (COVID-19) pandemic evolves and vaccine rollout progresses, the availability and demand for monoclonal antibodies for the prevention and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are also accelerating. This longitudinal serological study evaluated the magnitude and potency of the endogenous antibody response to COVID-19 vaccination in participants who first received a COVID-19 monoclonal antibody in a prevention study. Over the course of 6 months, serum samples were collected from a population of nursing home residents and staff enrolled in a clinical trial who were randomized to either bamlanivimab treatment or placebo. In an unplanned component of this trial, a subset of these participants was subsequently fully vaccinated with two doses of either SpikeVax (Moderna) or Comirnaty (BioNTech/Pfizer) COVID-19 mRNA vaccines. This post hoc analysis assessed the immune response to vaccination for 135 participants without prior SARS-CoV-2 infection. Antibody titers and potency were assessed using three assays against SARS-CoV-2 proteins that bamlanivimab does not efficiently bind to, thereby reflecting the endogenous antibody response. All bamlanivimab and placebo recipients mounted a robust immune response to full COVID-19 vaccination, irrespective of age, risk category, and vaccine type with any observed differences of uncertain clinical importance. These findings are pertinent for informing public health policy with results that suggest that the benefit of receiving COVID-19 vaccination at the earliest opportunity outweighs the minimal effect on the endogenous immune response due to prior prophylactic COVID-19 monoclonal antibody infusion.

Caspase-8 Variant G Regulates Rheumatoid Arthritis Fibroblast-Like Synoviocyte Aggressive Behavior.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) play a pivotal role in rheumatoid arthritis (RA) by contributing to synovial inflammation and progressive joint damage. An imprinted epigenetic state is associated with the FLS aggressive phenotype. We identified CASP8 (encoding for caspase-8) as a differentially marked gene and evaluated its pathogenic role in RA FLSs. METHODS: RA FLS lines were obtained from synovial tissues at arthroplasty and used at passage 5-8. Caspase-8 was silenced using small interfering RNA, and its effect was determined in cell adhesion, migration and invasion assays. Quantitative reverse transcription PCR and western blot were used to assess gene and protein expression, respectively. A caspase-8 selective inhibitor was used determine the role of enzymatic activity on FLS migration and invasion. Caspase-8 isoform transcripts and epigenetic marks in FLSs were analyzed in FLS public databases. Crystal structures of caspase-8B and G were determined. RESULTS: Caspase-8 deficiency in RA FLSs reduced cell adhesion, migration, and invasion independent of its catalytic activity. Epigenetic and transcriptomic analyses of RA FLSs revealed that a specific caspase-8 isoform, variant G, is the dominant isoform expressed (~80% of total caspase-8) and induced by PDGF. The crystal structures of caspase-8 variant G and B were identical except for a unique unstructured 59 amino acid N-terminal domain in variant G. Selective knockdown of caspase-8G was solely responsible for the effects of caspase-8 on calpain activity and cell invasion in FLS. CONCLUSION: Blocking caspase-8 variant G could decrease cell invasion in diseases like RA without the potential deleterious effects of nonspecific caspase-8 inhibition.

ImmUniverse Consortium: Multi-omics integrative approach in personalized medicine for immune-mediated inflammatory diseases.

Immune mediated inflammatory diseases (IMIDs) are a heterogeneous group of debilitating, multifactorial and unrelated conditions featured by a dysregulated immune response leading to destructive chronic inflammation. The immune dysregulation can affect various organ systems: gut (e.g., inflammatory bowel disease), joints (e.g., rheumatoid arthritis), skin (e.g., psoriasis, atopic dermatitis), resulting in significant morbidity, reduced quality of life, increased risk for comorbidities, and premature death. As there are no reliable disease progression and therapy response biomarkers currently available, it is very hard to predict how the disease will develop and which treatments will be effective in a given patient. In addition, a considerable proportion of patients do not respond sufficiently to the treatment. ImmUniverse is a large collaborative consortium of 27 partners funded by the Innovative Medicine Initiative (IMI), which is sponsored by the European Union (Horizon 2020) and in-kind contributions of participating pharmaceutical companies within the European Federation of Pharmaceutical Industries and Associations (EFPIA). ImmUniverse aims to advance our understanding of the molecular mechanisms underlying two immune-mediated diseases, ulcerative colitis (UC) and atopic dermatitis (AD), by pursuing an integrative multi-omics approach. As a consequence of the heterogeneity among IMIDs patients, a comprehensive, evidence-based identification of novel biomarkers is necessary to enable appropriate patient stratification that would account for the inter-individual differences in disease severity, drug efficacy, side effects or prognosis. This would guide clinicians in the management of patients and represent a major step towards personalized medicine. ImmUniverse will combine the existing and novel advanced technologies, including multi-omics, to characterize both the tissue microenvironment and blood. This comprehensive, systems biology-oriented approach will allow for identification and validation of tissue and circulating biomarker signatures as well as mechanistic principles, which will provide information about disease severity and future disease progression. This truly makes the ImmUniverse Consortium an unparalleled approach.

A humanized CD3ε-knock-in mouse model for pre-clinical testing of anti-human CD3 therapy.

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies.

Endogenous Antibody Responses to SARS-CoV-2 in Patients With Mild or Moderate COVID-19 Who Received Bamlanivimab Alone or Bamlanivimab and Etesevimab Together.

Background: Neutralizing monoclonal antibodies (mAbs) to SARS-CoV-2 are clinically efficacious when administered early, decreasing hospitalization and mortality in patients with mild or moderate COVID-19. We investigated the effects of receiving mAbs (bamlanivimab alone and bamlanivimab and etesevimab together) after SARS-CoV-2 infection on the endogenous immune response. Methods: Longitudinal serum samples were collected from patients with mild or moderate COVID-19 in the BLAZE-1 trial who received placebo (n=153), bamlanivimab alone [700 mg (n=100), 2800 mg (n=106), or 7000 mg (n=98)], or bamlanivimab (2800 mg) and etesevimab (2800 mg) together (n=111). A multiplex Luminex serology assay measured antibody titers against SARS-CoV-2 antigens, including SARS-CoV-2 protein variants that evade bamlanivimab or etesevimab binding, and SARS-CoV-2 pseudovirus neutralization assays were performed. Results: The antibody response in patients who received placebo or mAbs had a broad specificity. Titer change from baseline against a receptor-binding domain mutant (Spike-RBD E484Q), as well as N-terminal domain (Spike-NTD) and nucleocapsid protein (NCP) epitopes were 1.4 to 4.1 fold lower at day 15-85 in mAb recipients compared with placebo. Neutralizing activity of day 29 sera from bamlanivimab monotherapy cohorts against both spike E484Q and beta variant (B.1.351) were slightly reduced compared with placebo (by a factor of 3.1, p=0.001, and 2.9, p=0.002, respectively). Early viral load correlated with the subsequent antibody titers of the native, unmodified humoral response (p<0.0001 at Day 15, 29, 60 and 85 for full-length spike). Conclusions: Patients with mild or moderate COVID-19 treated with mAbs develop a wide breadth of antigenic responses to SARS-CoV-2. Small reductions in titers and neutralizing activity, potentially due to a decrease in viral load following mAb treatment, suggest minimal impact of mAb treatment on the endogenous immune response.

Generation and Characterization of Torudokimab (LY3375880): A Monoclonal Antibody That Neutralizes Interleukin-33.

BACKGROUND: Interleukin-33 (IL-33) is an alarmin that is released following cellular damage, mechanical injury, or necrosis. It is a member of the IL-1 family and binds to a heterodimer receptor consisting of ST2 and IL-1RAP to induce the production of a wide range of cellular mediators, including the type 2 cytokines IL-4, IL-5 and IL-13. This relationship has led to the hypothesis that the IL-33/ST2 pathway is a driver of allergic disease and inhibition of the IL-33 and ST2 association could have therapeutic benefit. METHODS: In this paper, we describe the selection of a phage antibody through the ability to bind human IL-33 and block IL-33/ST2 interaction. This hit antibody was then affinity matured by site-directed mutagenesis of the antibody complementarity-determining regions (CDRs). Further characterization of a fully human monoclonal antibody (mAb), torudokimab (LY3375880) included demonstration of human IL-33 neutralization activity in vitro with an NFκB reporter assay and IL-33 induced mast cell cytokine secretion assay, followed by an in vivo IL-33-induced pharmacodynamic inhibition assay in mice that used IL-5 production as the endpoint. RESULTS: Torudokimab is highly specific to IL-33 and does not bind any of the other IL-1 family members. Furthermore, torudokimab binds human and cynomolgus monkey IL-33 with higher affinity than the binding affinity of IL-33 to ST2, but does not bind mouse, rat, or rabbit IL-33. Torudokimab's half-life in cynomolgous monkey projects monthly dosing in the clinic. CONCLUSION: Due to torudokimab's high affinity, its ability to completely neutralize IL-33 activity in vitro and in vivo, and the observed cynomolgus monkey pharmacokinetic properties, this molecule was selected for clinical development.