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.

Structural features underlying raloxifene's biophysical interaction with bone matrix.

Raloxifene, a selective estrogen receptor modulator (SERM), reduces fracture risk at least in part by improving the mechanical properties of bone in a cell- and estrogen receptor-independent manner. In this study, we determined that raloxifene directly interacts with the bone tissue. Through the use of multiple and complementary biophysical techniques including nuclear magnetic resonance (NMR) and Fourier transform infrared spectroscopy (FTIR), we show that raloxifene interacts specifically with the organic component or the organic/mineral composite, and not with hydroxyapatite. Structure-activity studies reveal that the basic side chain of raloxifene is an instrumental determinant in the interaction with bone. Thus, truncation of portions of the side chain reduces bone binding and also diminishes the increase in mechanical properties. Our results support a model wherein the piperidine interacts with bone matrix through electrostatic interactions with the piperidine nitrogen and through hydrophobic interactions (van der Waals) with the aliphatic groups in the side chain and the benzothiophene core. Furthermore, in silico prediction of the potential binding sites on the surface of collagen revealed the presence of a groove with sufficient space to accommodate raloxifene analogs. The hydroxyl groups on the benzothiophene nucleus, which are necessary for binding of SERMs to the estrogen receptor, are not required for binding to the bone surface, but mediate a more robust binding of the compound to the bone powder. In conclusion, we report herein a novel property of raloxifene analogs that allows them to interact with the bone tissue through potential contacts with the organic matrix and in particular collagen.

2023 White Paper on Recent Issues in Bioanalysis: ISR for ADA Assays, the Rise of dPCR vs qPCR, International Reference Standards for Vaccine Assays, Anti-AAV TAb Post-Dose Assessment, NanoString Validation, ELISpot as Gold Standard (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Biotherapeutics Immunogenicity & Risk Assessment; ADA/NAb Assay/Reporting Harmonization).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) are published in volume 16 of Bioanalysis, issues 8 and 9 (2024), respectively.

Upfront acid treatment with low final pH: the development and testing of a novel method to improve drug tolerance in antidrug antibody assays.

Background: The drug tolerance of an antidrug antibody (ADA) assay for a therapeutic monoclonal antibody was insufficient to meet the level of biotherapeutic expected in sera, and a typical acid dissociation method was inadequate. Other strategies were investigated to dissociate ADA-drug complexes and thereby improve drug tolerance. Results: Having a lower final pH of samples after acid dissociation was shown to greatly improve drug tolerance. This method was shown to improve drug tolerance in the ADA assays for four additional monoclonal antibodies and to better detect ADAs in clinical samples. Conclusion: These findings provide a novel alternative method for improving drug tolerance when other methods are not sufficient.

A sensitive and drug tolerant assay for detecting anti-AAV9 antibodies using affinity capture elution.

Adeno-associated virus (AAV) based gene therapies are gaining significant momentum as a novel therapeutic modality. However, a yet unsolved concern for using AAV as a vector is the high potential to elicit humoral and cellular responses, which are often exacerbated by pre-existing immunity due to exposure to wild type AAV. Therefore, characterization of pre-existing and treatment emergent anti-AAV antibodies is of great importance to the development of AAV based gene therapies. In this project, a sensitive and drug tolerant total antibody (TAb) assay was developed using recombinant AAV9-GFP (green fluorescent protein) as a surrogate AAV9. The assay format was affinity capture and elution (ACE) with ruthenium labeled AAV9-GFP as detection. Upon evaluation, three commercial anti-AAV9 monoclonal antibodies (clones HI17, HI35, and HL2374) were chosen and mixed at equal concentrations as positive control material. The assay sensitivity was estimated to be 11.2 ng/mL. Drug tolerance was estimated to be 5.4 × 10E10 DRP/mL AAV9-GFP at 100 ng/mL anti-AAV9 antibodies and to be at least 1 × 10E11 DRP/mL at 500 ng/mL and 250 ng/mL anti-AAV9 antibodies. The assay showed desirable specificity and precision. Using this TAb assay, significant pre-existing antibodies were detected from normal human sera.

A Monovalent Fab Affinity-Capture and Elution Bridging Immunoassay Overcomes Rheumatoid Factor Interference while Accurately Detecting Antidrug Antibodies.

BACKGROUND: Rheumatoid factor (RF) consists of autoantibodies that bind the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) and present in sera of rheumatoid arthritis (RA) patients. Immunoassays to detect antidrug antibodies (ADA) in RA patient samples may experience interference due to RF binding and crosslinking Fc regions of the capture and detection antibody reagents. To overcome this interference, a novel Fab affinity-capture and elution (ACE)-bridging immunoassay (Fab ACE-Bridge) was developed with monovalent-recombinant Fab to avoid RF interference. METHODS: ACE and ACE-Bridge assays were developed to detect ADA against a therapeutic monoclonal antibody using samples from healthy donors, psoriasis patients, and RA patients. The performance of these assays was compared to a novel Fab ACE-Bridge assay, in which monoclonal antibody was replaced with monovalent Fab. RESULTS: High screening signals in the ACE and ACE-Bridge assays were detected in RA patient samples but not in samples from healthy donors or psoriasis patients. The high screening signals in RA samples did not inhibit to the expected extent in the confirmatory assay, a consistent feature of false-positive screening results. Further investigation revealed RF as the interferent affecting assay performance. Modification of the ACE-Bridge assay by using monovalent Fab eliminated RF interference while allowing for sensitive and drug-tolerant detection of authentic ADA. CONCLUSIONS: RF interfered significantly in traditional ACE and ACE-Bridge assays. Implementation of a novel monovalent Fab ACE-Bridge assay overcame RF interference. The use of monovalent Fab is recommended for immunogenicity assays when assessing ADA in RA patient samples.

Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

2022 White Paper on Recent Issues in Bioanalysis: FDA Draft Guidance on Immunogenicity Information in Prescription Drug Labeling, LNP & Viral Vectors Therapeutics/Vaccines Immunogenicity, Prolongation Effect, ADA Affinity, Risk-based Approaches, NGS, qPCR, ddPCR Assays (Part 3 - Recommendations on Gene Therapy, Cell Therapy, Vaccines Immunogenicity & Technologies; Immunogenicity & Risk Assessment of Biotherapeutics and Novel Modalities; NAb Assays Integrated Approach).

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26-30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

Deletion of Cx43 from osteocytes results in defective bone material properties but does not decrease extrinsic strength in cortical bone.

Deletion of connexin (Cx) 43 from osteoblasts and osteocytes (OCN-Cre;Cx43(fl/-) mice) or from osteocytes only (DMP1-8kb-Cre;Cx43(fl/fl) mice) results in increased cortical, but not cancellous, osteocyte apoptosis and widening of the femoral midshaft without changes in cortical thickness. Despite the consequent larger moment of inertia, stiffness and ultimate load, measures of mechanical strength assessed by three-point bending, are not higher in either model of Cx43 deficiency due to reduced Young's modulus, a measure of the stiffness of the material per unit of area. In OCN-Cre;Cx43(fl/-) mice, this was accompanied by a reduced ratio of nonreducible/reducible collagen cross-links as assessed by Fourier transformed infrared imaging (FTIRI) in the femoral diaphysis. On the other hand, DMP1-8kb-Cre;Cx43(fl/fl) mice did not show a significant reduction in collagen maturation in the same skeletal site, but a small decrease in mineralization was detected by FTIRI. Remarkably, both osteoblastic and osteocytic cells lacking Cx43 expressed lower mRNA levels of lysyl oxidase, a crucial enzyme involved in collagen maturation. These findings suggest that Cx43 expression in osteoblasts is involved in maintaining the quality of the bone matrix in cortical bone through the maturation of collagen cross-links. Osteocytic Cx43 expression is important also to maintain the stiffness of the bone material, where Cx43 deficiency results in local reduction in mineralization, possibly due to osteocyte apoptosis.

Connexin43 interacts with ßarrestin: a pre-requisite for osteoblast survival induced by parathyroid hormone.

Parathyroid hormone (PTH) promotes osteoblast survival through a mechanism that depends on cAMP-mediated signaling downstream of the G protein-coupled receptor PTHR1. We present evidence herein that PTH-induced survival signaling is impaired in cells lacking connexin43 (Cx43). Thus, expression of functional Cx43 dominant negative proteins or Cx43 knock-down abolished the expression of cAMP-target genes and anti-apoptosis induced by PTH in osteoblastic cells. In contrast, cells lacking Cx43 were still responsive to the stable cAMP analog dibutyril-cAMP. PTH survival signaling was rescued by transfecting wild type Cx43 or a truncated dominant negative mutant of ßarrestin, a PTHR1-interacting molecule that limits cAMP signaling. On the other hand, Cx43 mutants lacking the cytoplasmic domain (Cx43(Δ245)) or unable to be phosphorylated at serine 368 (Cx43(S368A)), a residue crucial for Cx43 trafficking and function, failed to restore the anti-apoptotic effect of PTH in Cx43-deficient cells. In addition, overexpression of wild type ßarrestin abrogated PTH survival signaling in Cx43-expressing cells. Moreover, ßarrestin physically associated in vivo to wild type Cx43 and to a lesser extent to Cx43(S368A) ; and this association and the phosphorylation of Cx43 in serine 368 were reduced by PTH. Furthermore, induction of Cx43(S368) phosphorylation or overexpression of wild type Cx43, but not Cx43(Δ245) or Cx43(S368A) , reduced the interaction between ßarrestin and the PTHR1. These studies demonstrate that ßarrestin is a novel Cx43-interacting protein and suggest that, by sequestering ßarrestin, Cx43 facilitates cAMP signaling, thereby exerting a permissive role on osteoblast survival induced by PTH.

Nmp4/CIZ suppresses the response of bone to anabolic parathyroid hormone by regulating both osteoblasts and osteoclasts.

How parathyroid hormone (PTH) increases bone mass is unclear, but understanding this phenomenon is significant to the improvement of osteoporosis therapy. Nmp4/CIZ is a nucleocytoplasmic shuttling transcriptional repressor that suppresses PTH-induced osteoblast gene expression and hormone-stimulated gains in murine femoral trabecular bone. To further characterize Nmp4/CIZ suppression of hormone-mediated bone growth, we treated 10-week-old Nmp4-knockout (KO) and wild-type (WT) mice with intermittent human PTH(1-34) at 30 µg/kg daily or vehicle, 7 days/week, for 2, 3, or 7 weeks. Null mice treated with hormone (7 weeks) gained more vertebral and tibial cancellous bone than WT animals, paralleling the exaggerated response in the femur. Interestingly, Nmp4/CIZ suppression of this hormone-stimulated bone formation was not apparent during the first 2 weeks of treatment. Consistent with the null mice enhanced PTH-stimulated addition of trabecular bone, these animals exhibited an augmented hormone-induced increase in serum osteocalcin 3 weeks into treatment. Unexpectedly, the Nmp4-KO mice displayed an osteoclast phenotype. Serum C-terminal telopeptide, a marker for bone resorption, was elevated in the null mice, irrespective of treatment. Nmp4-KO bone marrow cultures produced more osteoclasts, which exhibited elevated resorbing activity, compared to WT cultures. The expression of several genes critical to the development of both osteoblasts and osteoclasts was elevated in Nmp4-KO mice at 2 weeks, but not 3 weeks, of hormone exposure. We propose that Nmp4/CIZ dampens PTH-induced improvement of trabecular bone throughout the skeleton by transiently suppressing hormone-stimulated increases in the expression of proteins key to the required enhanced activity and number of both osteoblasts and osteoclasts.

Novel methodology comparing two assay formats for the assessment of immunogenicity from clinical trial samples.

Aim: We present a novel methodology to compare results between distinct immunogenicity assays, performed by two laboratories, for the same biotherapeutic. Materials & methods: Human serum pools from clinical trials were generated to provide representative immunogenicity titers. Pools were evaluated at two laboratories in a blinded fashion to assess the effect of assay format and laboratory change on clinical interpretation of immunogenicity results. Results: The laboratories validated two different assay formats and demonstrated comparable sensitivity and drug tolerance. Overall, the comparisons in assay format and laboratory ensured a comparable ability to detect treatment-emergent antidrug antibodies for a biotherapeutic. Conclusion: We have established an approach, using pooling of patient samples, that allows for the interlaboratory comparisons without creating duplicative results.

Lay abstract Measuring immunogenicity, an immune response to a drug, is important in understanding the benefits and risks associated with a drug. Immunogenicity is measured by specific tests within a laboratory; however, these tests and laboratories may change over time. This paper proposes a method to determine if a change in test and laboratory will impact the interpretation of immunogenicity for a drug. Blood samples from clinical trial patients were combined in order to provide representative samples for the immunogenicity tests. The samples were tested at two laboratories with two tests to measure if any interpretation of immunogenicity results would change due to the different tests and laboratories. Laboratories and tests demonstrated similar and reliable results of the samples. This study has established a method which allows for the comparison of immunogenicity results when tests and/or laboratories are changed.

Safety and Immunogenicity of LY3415244, a Bispecific Antibody Against TIM-3 and PD-L1, in Patients With Advanced Solid Tumors.

PURPOSE: Investigate the safety and efficacy of LY3415244, a TIM-3/PD-L1 bispecific antibody that blocks TIM-3 and PD-L1 in patients with advanced solid tumors. PATIENTS AND METHODS: A phase I, multicenter, open-label study was conducted in patients with advanced solid tumors. Patients were dosed every 2 weeks intravenously with flat doses of LY3415244 escalating from 3 to 70 mg. The primary endpoints were safety, tolerability, and identification of the recommended phase II dose. RESULTS: Between November 2018 and October 2019, 12 patients were enrolled into four cohorts and received at least one dose of LY3415244. Two patients (16.7%) developed clinically significant anaphylactic infusion-related reactions and all patients developed treatment-emergent antidrug antibodies (TE-ADA). ADA titers were sometimes very high and negatively impacted soluble TIM-3 target engagement in most patients. ADA epitope specificity was against both TIM-3 and PD-L1 arms of the bispecific antibody; most TE-ADAs initially targeted the TIM-3 arm after the first dose. Preexisting ADAs against LY3415244 were also detected in normal (unexposed) human serum samples. One patient with PD-1 refractory non-small cell lung cancer had a near partial response (-29.6%). CONCLUSIONS: This TIM-3 and PD-L1 bispecific format was associated with unexpected immunogenicity targeting both arms of the bispecific antibody, resulting in early study termination. Epitope specificity analysis revealed an initial response toward the TIM-3 arm and presence of preexisting ADAs to the bispecific molecule in the general population. This experience emphasizes the importance of thorough analyses for preexisting ADAs as part of immunogenicity risk assessment of novel antibodies.See related commentary by de Spéville and Moreno, p. 2669.

Development and validation of a novel immunogenicity assay to detect anti-drug and anti-PEG antibodies simultaneously with high sensitivity.

Polyethylene glycol (PEG) represents an effective strategy to improve the pharmacokinetic profile of a molecule as it extends the biotherapeutic's half-life, masks immunogenic epitopes or modifies its distribution. The addition of one or multiple PEG moieties, in either linear or branched form, is known to carry the risk of potentially inducing an immunogenic response against PEG. The importance of accurately quantifying anti-PEG antibodies during a clinical study is well recognized and stems from the fact that anti-PEG antibodies have been shown to negatively impact the efficacy of the biotherapeutic that the PEG is coupled to. As a consequence, sponsors are encouraged to develop immunogenicity assays to assess appropriately the presence of anti-drug antibodies (ADA) against the protein component as well as the PEG. However, detection of anti-PEG antibodies is complicated by a number of technical challenges, including the availability of appropriate positive control material. In addition, the fact that some anti-PEG antibodies are known to circulate as low-affinity IgM, drives the need for an assay able to detect low affinity anti-PEG ADA even in the presence of high concentrations of the biotherapeutic. To address this need, we developed and validated an Affinity Capture Elution (ACE)-AGL assay to detect anti-drug and anti-PEG antibodies. In this assay, which we call ACE-AGL, ADA are captured by biotin-PEG-drug, acid eluted and re-captured on a second plate coated with protein AGL. ADA are then detected using Ruthenium-PEG-drug. The new assay format described is highly sensitive to both anti-drug and anti-PEG antibodies and very drug-tolerant. The ACE-AGL assay is easy to perform and has been successfully validated at two separate CROs. We propose the ACE-AGL format as a valid and effective alternative to the currently available assay methods.

The method history report: an adaptable tool for communicating immunogenicity assay development and validation.

Over the developmental lifetime of a therapeutic protein, the immunogenicity assay validation history can become substantial, frustrating review of clinical immunogenicity within the biologics license application. In our experience, this can lead to questions by regulators, resulting in numerous information requests during the review process. To address this, we propose a new document, the method history report (MHR), which can comprehensively present the history of the immunogenicity assay for regulators, including assay development and validation. The flexibility of the MHR allows for adaptation to the specific needs of each therapeutic program, while maintaining a consistent template. Here, we detail the rationale, general outline and template for the MHR and recommend others consider adopting it for their biologics license application-related activities.

Stat3 in osteocytes mediates osteogenic response to loading.

Signal transducer and activator of transcription 3 (Stat3) is a member of the Stat family of proteins involved in signaling in many different cell types, including osteocytes. Osteocytes are considered major mechanosensing cells in bone due to their intricate dendritic networks able to sense changes in physical force and to orchestrate the response of osteoclasts and osteoblasts. We examined the role of Stat3 in osteocytes by generating mice lacking Stat3 in these cells using the Dmp-1(8kb)-Cre promoter (Stat3cKO mice). Compared to age-matched littermate controls, Stat3cKO mice of either sex (18 weeks old) exhibit reduced bone formation indices, decreased osteoblasts and increased osteoclasts, and altered material properties, without detectable changes in bone mineral density (BMD) or content of either trabecular or cortical bone. In addition, Stat3cKO mice of either sex show significantly decreased load-induced bone formation. Furthermore, pharmacologic inhibition of Stat3 in osteocytes in vitro with WP1066 blocked the increase in cytosolic calcium induced by ATP, a mediator of the cellular responses to sheer stress. WP1066 also increased reactive oxygen species (ROS) production in cultured MLO-Y4 osteocytes. These data demonstrate that Stat3 is a critical mediator of mechanical signals received by osteocytes and suggest that osteocytic Stat3 is a potential therapeutic target to stimulate bone anabolism.

Assessment and Clinical Relevance of Serum IL-19 Levels in Psoriasis and Atopic Dermatitis Using a Sensitive and Specific Novel Immunoassay.

Because development of reliable biomarkers in psoriasis and atopic dermatitis has lagged behind therapeutic progress, we created a blood-based test to fill the void in objective methods available for dermatological assessments. Our novel interleukin-19 (IL-19) immunoassay was initially tested to determine concentrations of IL-19 serum levels, then correlated with the psoriasis activity and severity index (PASI) in psoriasis, and the eczema area and severity index (EASI) in atopic dermatitis. Not only was IL-19 increased in psoriasis and correlated to PASI, but ixekizumab administration led to rapid, sustained IL-19 decreases to normal levels, with decreases at 2-weeks correlating with PASI improvement at 16-weeks. IL-19 increased upon ixekizumab withdraw, prior to relapse, and decreased following re-treatment. In baricitinib- and etanercept-treated psoriasis patients, IL-19 decreases also correlated with improvement. Many patients with limited skin disease, including genital psoriasis and psoriatic arthritis patients, also had increased IL-19, which was reduced to normal levels upon ixekizumab treatment, correlating with PASI improvement. We also measured IL-19 in baricitinib-treated atopic dermatitis patients. In atopic dermatitis, IL-19 was significantly elevated, correlated with EASI scores, and decreased with skin improvement. Therefore, measurement of serum IL-19 provides clinicians with an objective disease-activity assessment tool for psoriasis and atopic dermatitis patients.

Investigation of pre-existing reactivity to biotherapeutics can uncover potential immunogenic epitopes and predict immunogenicity risk.

Despite recent advances in the development of tools to predict immunogenicity risk of biotherapeutic molecules, the ability of a protein to elicit the formation of anti-drug antibodies (ADA) remains one of the most common causes for termination of clinical development programs. In this study, we use ADA assays to detect and measure pre-existing reactivity or the ability of a molecule to produce an ADA-like response in serum from treatment-naïve, healthy donors. We report herein that the magnitude of pre-existing reactivity evaluated pre-clinically and expressed as the 90th percentile of Tier 2 inhibition correlates with the subsequent rate of ADA emergence in the clinic. Furthermore, a multi-domain biotherapeutic (IgG-scFv bispecific antibody) showed the highest pre-existing reactivity and incidence of treatment-emergent ADA (TE-ADA) (57% and 93%, respectively). Using the components of the multidomain molecule in the Tier 2 step of the ADA assay, we were able to identify the scFv as the target of the serum pre-existing reactivity. Most importantly, the domain specificity of pre-existing ADA was the same as that of the TE-ADA from patients treated with the molecule. Based on these data, we propose the evaluation of the magnitude and of the domain specificity of pre-existing reactivity as a powerful tool to understand the immunogenic potential of novel biotherapeutics.

APE1/Ref-1 regulates PTEN expression mediated by Egr-1.

APE1/Ref-1, the mammalian ortholog of E. coli Xth, and a multifunctional protein possessing both DNA repair and transcriptional regulatory activities, has dual role in controlling cellular response to oxidative stress. It is rate-limiting in repair of oxidative DNA damage including strand breaks and also has co-transcriptional activity by modulating genes expression directly regulated by Egr-1 and p53 transcription factors. PTEN, a phosphoinositide phosphatase, acts as an 'off' switch in the PI-3 kinase/Akt signalling pathway and regulates cell growth and survival. It is shown here that transient alteration in the APE1 level in HeLa cells modulates PTEN expression and that acetylatable APE1 is required for the activation of the PTEN gene. Acetylation of APE1 enhances its binding to distinct trans-acting complexes involved in activation or repression. The acetylated protein is deacetylated in vivo by histone deacetylases. It was found that exposure of HeLa cells to H(2)O(2) and to histone deacetylase inhibitors increases acetylation of APE1 and induction of PTEN. The absence of such induction in APE1-downregulated HeLa cells confirmed APE1's role in regulating inducible PTEN expression. That APE1-dependent PTEN expression is mediated by Egr-1 was supported by experiments with cells ectopically expressing Egr-1. Thus, the data open new perspectives in the comprehension of the many functions exerted by APE1 in controlling cell response to oxidative stress.