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Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

Some health concerns are often not identified until late into clinical development of drugs, which can place participants and patients at significant risk. For example, the United States Food and Drug Administration (FDA) labeled the xanthine oxidase inhibitor febuxostat with a”boxed” warning regarding an increased risk of cardiovascular death, and this safety risk was only identified during Phase 3b clinical trials after its approval. Thus, better preclinical assessment of drug efficacy and safety are needed to accurately evaluate candidate drug risk earlier in discovery and development. This study explored whether an in vitro vascular model incorporating human vascular cells and hemodynamics could be used to differentiate the potential cardiovascular risk associated with molecules that have similar on-target mechanisms of action. We compared the transcriptomic responses induced by febuxostat and other xanthine oxidase inhibitors to a database of 111 different compounds profiled in the human vascular model. Of the 111 compounds in the database, 107 are clinical-stage and 33 are FDA-labelled for increased cardiovascular risk. Febuxostat induces pathway-level regulation that has high similarity to the set of drugs FDA-labelled for increased cardiovascular risk. These results were replicated with a febuxostat analog, but not another structurally distinct xanthine oxidase inhibitor that does not confer cardiovascular risk. Together, these data suggest that the FDA warning for febuxostat stems from the chemical structure of the medication itself, rather than the target, xanthine oxidase. Importantly, these data indicate that cardiovascular risk can be evaluated in this in vitro human vascular model, which may facilitate understanding the drug candidate safety profile earlier in discovery and development.

Immunoglobulin E （IgE） plays a key role in allergic asthma and is a clinically validated target for monoclonal antibodies. Therapeutic anti-lgE antibodies block the interaction between IgE and the Fc epsilon （Fcε） receptor, which eliminates or minimizes the allergic phenotype but does not typically curtail the ongoing production of IgE by B cells. We generated high-affinity anti-lgE antibodies （MEDI4212） that have the potential to both neutralize soluble IgE and eliminate IgE-expressing B-cells through antibody-dependent cell-mediated cytotoxicity. MEDI4212 variants were generated that contain mutations in the Fc region of the antibody or alterations in fucosylation in order to enhance the antibody＇s affinity for FcyRIIla. All MEDI4212 variants bound to human IgE with affinities comparable to the wild-type （WT） antibody. Each variant was shown to inhibit the interaction between IgE and FcεRI, which translated into potent inhibition of FcyRI-mediated function responses. Importantly, all variants bound similarly to IgE at the surface of membrane IgE expressing cells. However, MEDI4212 variants demonstrated enhanced affinity for FcyRIIla including the polymorphic variants at position 158. The improvement in FcyRIIla binding led to increased effector function in cell based assays using both engineered cell lines and class switched human IgE B cells. Through its superior suppression of IgE, we anticipate that effector function enhanced MEDI4212 may be able to neutralize high levels of soluble IgE and provide increased long-term benefit by eliminating the IgE expressing B cells before they differentiate and become IgE secreting plasma cells.

Azotobacter vinelandii flavodoxin hydroquinone (FIdHQ) is a physiological reductant to nitrogenase supporting catalysis that is twice as energy efficient (ATP/2e⁻ = 2) as dithionite (ATP/2e⁻ = 4). This catalytic efficiency results from reduction of Fe protein from A. vinelandii (Av2) to the all-ferrous oxidation state ([Fe₄S₄]⁰), in contrast to dithionite, which only reduces Av2 to the [Fe₄S₄]¹⁺ state. Like FIdHQ, Ti(III) citrate yields ATP/2e⁻ = 2, and Ti(III)reduced [Fe₄S₄]⁰ Av2 has a S = 4 spin state and characteristic Mossbauer spectrum, a parallel mode g = 16.4 EPR signal, and a shoulder at 520 nm in its UV-vis spectrum, each of which distinguish the S = 4 [Fe₄S₄]⁰ Av2 from other states. In this study, we demonstrate that FIdHQ makes [Fe₄S₄]⁰ Av2, which is sufficiently characterized to demonstrate unique physical properties that distinguish it from the previously characterized Ti(III)-reduced [Fe₄S₄]⁰ Av2. In particular, Evans NMR magnetic susceptibility and EPR measurements indicate that FIdHQ-reduced [Fe₄S₄]⁰ Av2 has an S = 0 spin state (like [Fe₄S₄]²⁺ Av2). There is no g = 16.4 EPR signal and no shoulder at 520 nm in its absorbance spectrum, which resembles that of [Fe₄S₄]¹⁺ Av2. That the physiological reductant to Av2 is capable of forming [Fe₄S₄]⁰ Av2 has important implications for in vivo nitrogenase activity.

Background The quadrivalent live attenuated influenza vaccine (LAIV4) showed reduced effectiveness for the A/H1N1 component of the vaccine in 2013–2014 and 2015–2016. To address this, new assays were used to identify H1N1 LAIV strains with improved replicative fitness and immunogenicity. In this study, we compared the shedding and immunogenicity of a new A/H1N1 strain (A/Slovenia), selected using the new assays, to a previous strain (A/Bolivia) with reduced effectiveness. Methods Two hundred children aged 24 to <48 months were randomized 1:1:1 to receive two doses of a quadrivalent formulation of 2015–16 LAIV or a trivalent formulation of 2015–2016 LAIV, both containing the H1N1 A/Bolivia strain, or a quadrivalent formulation of the 2017–2018 LAIV containing the new H1N1 A/Slovenia strain (NCT03143101). Nasal and serum immune responses were assessed before Doses 1 and 2, and 28 days after Dose 2. Nasal shedding was assessed on Days 2, 3, 4, 5, and 7 after Dose 1, and Days 2, 4, and 6 after Dose 2. Solicited symptoms, adverse events, and serious adverse events were collected. Statistical testing was limited to the prespecified primary endpoint of hemagglutination inhibition (HAI) antibody responses. Results A higher proportion of children shed the A/Slovenia vaccine strain than the A/Bolivia strain on Days 4–7 after Dose 1. The study met its primary endpoint, with significantly higher HAI antibody responses for the A/Slovenia strain after both the first and second doses of vaccine (Figure 1). Neutralizing antibodies and nasal immunoglobulin A (IgA) antibody responses were higher for the A/Slovenia than the A/Bolivia strain for both the trivalent and quadrivalent vaccine formulations. HAI antibody seroconversion rates (≥4-fold increase from baseline) for the A/Slovenia strain were similar to those seen in previous studies in which the H1N1 vaccine strain was highly efficacious (Figure 2). There were no significant safety findings. Conclusion The new H1N1 A/Slovenia strain demonstrated improved immunogenicity compared with a previous strain with reduced effectiveness, and immune responses comparable to a highly efficacious H1N1 LAIV strain. These results support the use of LAIV4 as an important vaccine option. Disclosures R. Mallory, MedImmune: Employee, Salary. A.C. Nyborg, MedImmune: Employee, Salary. R. Kalyani, MedImmune: Employee, Salary. L.F. Tsai, MedImmune: Employee, Salary. S.L. Block, AstraZeneca: Investigator, Research grant. F. Dubovsky, MedImmune: Employee, Salary.

Abstract Background Influenza is responsible for significant morbidity and mortality and more effective therapeutics are needed. MEDI8852 is a human IgG1 kappa monoclonal antibody that binds to the conserved stalk region of the influenza hemagglutinin protein neutralizing both seasonal and pandemic influenza A strains. MEDI8852 is being developed to treat patients hospitalized with influenza A. The primary objective of this study was to evaluate the safety of MEDI8852 administered either alone or with oseltamivir (OS) in outpatient adults with acute, uncomplicated influenza A. Methods Subjects aged 18 to 65 years with onset of influenza symptoms ≤ 5 days prior to dosing were enrolled. Subjects were randomized into 4 cohorts: Cohort 1: 750 mg MEDI8852 (single intravenous infusion) + 75 mg OS (orally twice a day for 5 days); Cohort 2: 3,000 mg MEDI8852 + 75 mg OS; Cohort 3: placebo + 75 mg OS; or Cohort 4: 3,000 mg MEDI8852. Subjects were followed through Day 10 for solicited symptoms (SS), through Day 28 for adverse events (AEs) and through Day 101 for serious AEs (SAEs) and AEs of special interest (AESIs). Viral shedding was assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) through Day 7. Results 126 subjects (31 in Cohort 1; 31 in Cohort 2; 32 in Cohort 3; and 32 in Cohort 4) were enrolled at sites in United States (2015–16) and South Africa (2016). More AEs were reported in the total MEDI8852 group (Cohorts 1, 2 and 4 combined: 39/93, 41.9%) than in placebo group (10/32, 31.3%). Related AEs were similar across the 2 groups (14/93, 15.1% total MEDI8852 group; 5/32, 15.6% placebo group). Most AEs were Grade 1 or Grade 2. The most common AE was bronchitis (11/93, 11.8%; 1/32, 3.1%). One subject in Cohort 2 had a related SAE (and AESI) of Grade 3 infusion-related reaction, which resolved with treatment. There were no deaths or discontinuations due to an AE. Median (range) time to resolution of SS were similar across groups (93/94, 111.3 [92.4, 130.8] hours; 32/32, 108.8 [71.2, 161.8] hours). Median (range) decreases in viral shedding (log10 viral copies/mL) were similar across groups (-3.6 [-6.2. 0.5]; -3.4 [-5.9, 0.9]). Conclusion MEDI8852 has an acceptable safety profile in adults with acute, uncomplicated influenza A. These results support the continued development of MEDI8852 for the treatment of influenza A. Disclosures O. Ali, MedImmune: Employee, Salary; T. Takas, MedImmune: Employee and Shareholder, Salary and stock; A. C. Nyborg, MedImmune: Employee, Salary; K. M. Jensen, MedImmune: Employee and Shareholder, Salary and stock; F. Dubovsky, MedImmune: Employee and Shareholder, Salary and stock; R. Mallory, MedImmune: Employee, Salary