Safety, Tolerability, and Pharmacokinetics of an Oral Small Molecule Inhibitor of IL-17A (LY3509754): A Phase I Randomized Placebo-Controlled Study.

For some patients with psoriasis, orally administered small molecule inhibitors of interleukin (IL)-17A may represent a convenient alternative to IL-17A-targeting monoclonal antibodies. This first-in-human study assessed the safety, tolerability, pharmacokinetics (PKs), and peripherally circulating IL-17A target engagement profile of single or multiple oral doses of the small molecule IL-17A inhibitor LY3509754 (NCT04586920). Healthy participants were randomly assigned to receive LY3509754 or placebo in sequential escalating single ascending dose (SAD; dose range 10-2,000 mg) or multiple ascending dose (MAD; dose range 100-1,000 mg daily for 14 days) cohorts. The study enrolled 91 participants (SAD, N = 51 and MAD, N = 40) aged 21-65 years (71% men). LY3509754 had a time to maximum concentration (Tmax) of 1.5-3.5 hours, terminal half-life of 11.4-19.1 hours, and exhibited dose-dependent increases in exposure. LY3509754 had strong target engagement, indicated by elevated plasma IL-17A levels within 12 hours of dosing. Four participants from the 400-mg (n = 1) and 1,000-mg (n = 3) MAD cohorts experienced increased liver transaminases or acute hepatitis (onset ≥ 12 days post-last LY3509754 dose), consistent with drug-induced liver injury (DILI). One case of acute hepatitis was severe, resulted in temporary hospitalization, and was classified as a serious adverse event. No adverse effects on other major organ systems were observed. Liver biopsies from three of the four participants revealed lymphocyte-rich, moderate-to-severe lobular inflammation. We theorize that the DILI relates to an off-target effect rather than IL-17A inhibition. In conclusion, despite strong target engagement and a PK profile that supported once-daily administration, this study showed that oral dosing with LY3509754 was poorly tolerated.

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.

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.

Level and Duration of IgG and Neutralizing Antibodies to SARS-CoV-2 in Children with Symptomatic or Asymptomatic SARS-CoV-2 Infection.

There are conflicting data about level and duration of Abs to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in children after symptomatic or asymptomatic infection. In this human population, we enrolled adults and children in a prospective 6-mo study in the following categories: 1) symptomatic, SARS-CoV-2 PCR+ (SP+; children, n = 8; adults, n = 16), 2) symptomatic, PCR-, or untested (children, n = 27), 3) asymptomatic exposed (children, n = 13), and 4) asymptomatic, no known exposure (children, n = 19). Neutralizing Abs (nAbs) and IgG Abs to SARS-CoV-2 Ags and spike protein variants were measured by multiplex serological assay. All SP+ children developed nAb, whereas 81% of SP+ adults developed nAb. Decline in the presence of nAb over 6 mo was not significant in symptomatic children (100 to 87.5%; p = 0.32) in contrast to adults (81.3 to 50.0%; p = 0.03). Among children with nAb (n = 22), nAb titers and change in titers over 6 mo were similar in symptomatic and asymptomatic children. In children and adults, nAb levels postinfection were 10-fold lower than those reported after SARS-CoV-2 mRNA vaccination. Levels of IgG Abs in children to SARS-CoV-2 Ags and spike protein variants were similar to those in adults. IgG levels to primary Ags decreased over time in children and adults, but levels to three spike variants decreased only in children. Children with asymptomatic or symptomatic SARS-CoV-2 infection develop nAbs that remain present longer than in adults but wane in titer over time and broad IgG Abs that also wane in level over time. However, nAb levels were lower postinfection than those reported after immunization.

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.

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.

First-in-Human Study of Bamlanivimab in a Randomized Trial of Hospitalized Patients With COVID-19.

Therapeutics for patients hospitalized with coronavirus disease 2019 (COVID-19) are urgently needed during the pandemic. Bamlanivimab is a potent neutralizing monoclonal antibody that blocks severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) attachment and entry into human cells, which could potentially lead to therapeutic benefit. J2W-MC-PYAA was a randomized, double-blind, sponsor unblinded, placebo-controlled, single ascending dose first-in-human trial (NCT04411628) in hospitalized patients with COVID-19. A total of 24 patients received either placebo or a single dose of bamlanivimab (700 mg, 2,800 mg, or 7,000 mg). The primary objective was assessment of safety and tolerability, including adverse events and serious adverse events, with secondary objectives of pharmacokinetic (PK) and pharmacodynamic analyses. Treatment-emergent adverse event (TEAE) rates were identical in the placebo and pooled bamlanivimab groups (66.7%). There were no apparent dose-related increases in the number or severity of TEAEs. There were no serious adverse events or deaths during the study, and no discontinuations due to adverse events. PKs of bamlanivimab is linear and exposure increased proportionally with dose following single i.v. administration. The half-life was ~ 17 days. These results demonstrate the favorable safety profile of bamlanivimab, and provided the initial critical evaluation of safety, tolerability, and PKs in support of the development of bamlanivimab in several ongoing clinical trials.

Measurement of IL-21 in human serum and plasma using ultrasensitive MSD S-PLEX® and Quanterix SiMoA methodologies.

IL-21 is a pleiotropic cytokine that plays a key role in modulating inflammatory responses, including the promotion of autoimmune diseases. Several groups have quantitated circulating levels of IL-21 in plasma and serum samples using various commercial ELISAs. We determined, however, that the most commonly used commercial assays in published literature were not specific or sensitive enough to detect levels of IL-21 in heparin plasma or serum from healthy human individuals. This finding prompted an effort to develop more specific and sensitive methods to quantitate IL-21 in complex biological matrices using proprietary anti-IL-21 antibodies with the Quanterix SiMoA platform and the Meso Scale Discovery (MSD) S-PLEX® format. Assays developed on both technology platforms were characterized in heparin plasma and serum using spike recoveries across a range of concentrations. Each method was able to detect sub-pg/mL levels of IL-21 (predicted Limit of Detection [LOD] of approximately 1.0 fg/mL for both the Quanterix SiMoA and MSD S-PLEX® platforms) which is 200-500 times lower than current commercial assays. Additionally we demonstrated that rheumatoid factor did not interfere with measuring IL-21 in the Quanterix SiMoA assay. Results obtained with the two new ultrasensitive assays showed a strong correlation (r = 0.9428; p < .0001). Additionally, IL-21 levels were significantly increased in samples from patients with Systemic Lupus Erythematosus (mean+/- SD: n = 14, 202.64 +/- 111.47 fg/mL, p = .0001 for Quanterix SiMoA and 275.4 +/- 174.66 fg/mL p = .0001 for MSD S-PLEX®) as well as in samples from patients with Sjögren's Syndrome (mean+/- SD: n = 11, 122.18 +/- 84.50 fg/mL, p = .0029 for Quanterix SiMoA and 183.64 +/- 153.00 fg/mL, p = .0082 for MSD S-PLEX®) when compared to healthy donors (mean+/- SD: n = 11, 38.1 +/- 27.8 fg/mL for Quanterix SiMoA and 58.1 +/- 30.7 fg/mL for MSD S-PLEX®). These ultrasensitive assays, for the first time, allow for the accurate quantitation of human IL-21 in heparin plasma and serum. In addition, these experiments also provide a direct comparison of the MSD S-PLEX® format and Quanterix SiMoA platform technologies, which may have broader implications to future application of these methods to evaluate low abundance proteins in complex biological matrices.

Root cause investigation of a viral contamination incident occurred during master cell bank (MCB) testing and characterization--a case study.

An adventitious agent contamination occurred during a routine 9 CFR bovine viral screening test at BioReliance for an Eli Lilly Chinese Hamster Ovary (CHO) cell-derived Master Cell Bank (MCB) intended for biological production. Scientists from the sponsor (Eli Lilly and Company) and the testing service company (BioReliance) jointly conducted a systematic investigation in an attempt to determine the root cause of the contamination. Our investigation resulted in the identification of the viral nature of the contaminant. Subsequent experiments indicated that the viral contaminant was a non-enveloped and non-hemadsorbing virus. Transmission electron microscopy (TEM) revealed that the viral contaminant was 25-30 nm in size and morphologically resembled viruses of the family Picornaviridae. The contaminant virus was readily inactivated when exposed to acidic pH, suggesting that the viral contaminant was a member of rhinoviruses. Although incapable of infecting CHO cells, the viral contaminant replicated efficiently in Vero cell with a life cycle of approximately 16 h. Our investigation provided compelling data demonstrating that the viral contaminant did not originate from the MCB. Instead, it was introduced into the process during cell passaging and a possible entry point was proposed. We identified the viral contaminant as an equine rhinitis A virus using molecular cloning and DNA sequencing. Finally, our investigation led us to conclude that the source of the viral contaminant was the equine serum added to the cell growth medium in the 9 CFR bovine virus test.