The Impact of Immunogenicity on the Pharmacokinetics, Efficacy, and Safety of Sotatercept in a Phase III Study of Pulmonary Arterial Hypertension.

Sotatercept, a soluble fusion protein comprising the extracellular domain of activin receptor type IIA linked to the Fc portion of human IgG1, is a first-in-class activin signaling inhibitor under development for the treatment of pulmonary arterial hypertension (PAH). We evaluated antidrug antibody (ADA) development and determined the effects of immunogenicity on the pharmacokinetics (PKs), efficacy, and safety of sotatercept in STELLAR, a multicenter, double-blind phase III trial (NCT04576988) wherein participants with PAH were randomized 1:1 to receive sotatercept (starting dose 0.3; target dose 0.7 mg/kg) or placebo subcutaneously every 3 weeks in combination with background therapies for ≤ 72 weeks. ADA-positive (ADA-POS) participants were identified and characterized for neutralizing antibodies (NAbs). PKs, efficacy, and safety were evaluated by ADA and NAb status. Of 162 evaluable participants, 42 (25.9%) were ADA-POS through week 24, of whom 11 (6.8%) were also NAb-POS. Median onset of ADAs was 3.29 weeks (interquartile range (IQR): 3.14-6.14), and median duration was 6 weeks (IQR: 3.14-17.86). No clinically meaningful differences were found across subgroups that were ADA-NEG, ADA-POS/NAb-NEG, and ADA-POS/NAb-POS, in terms of PKs (sotatercept trough concentration over time, mean postdose trough concentration at the end of treatment, and clearance), efficacy (changes from baseline in 6-minute walk distance, pulmonary vascular resistance, and N-terminal pro-B-type natriuretic peptide levels), and safety (incidence of hypersensitivity, anaphylactic reactions, and administration site reactions). We conclude that ADA incidence from sotatercept treatment was 25.9% and did not meaningfully affect the PKs, efficacy, or safety of sotatercept in participants with PAH.

Roux-en-Y and One-Anastomosis Gastric Bypass Surgery Are Superior to Sleeve Gastrectomy in Lowering Glucose and Cholesterol Levels Independent of Weight Loss: a Propensity-Score Weighting Analysis.

BACKGROUND: The superior effects of gastric bypass surgery in preventing cardiovascular diseases compared with sleeve gastrectomy are well-established. However, whether these effects are independent of weight loss is not known. METHODS: In this retrospective cohort study, we compared the change in cardiometabolic risks of 1073 diabetic patients undergoing Roux-en-Y gastric bypass (RYGB) (n = 265), one-anastomosis gastric bypass (OAGB) (n = 619), and sleeve gastrectomy (SG) (n = 189) with equivalent weight loss from the Min-Shen General Hospital. Propensity score-weighting, multivariate regression, and matching were performed to adjust for baseline differences. RESULTS: After 12 months, OAGB and, to a lesser extent, RYGB exhibited superior effects on glycemic control compared with SG in patients with equivalent weight loss. The effect was significant in patients with mild-to-modest BMI reduction but diminished in patients with severe BMI reduction. RYGB and OAGB had significantly greater effects in lowering total and low-density lipoprotein cholesterol than SG, regardless of weight loss. The results of matching patients with equivalent weight loss yielded similar results. The longer length of bypassed biliopancreatic (BP) limbs was correlated with a greater decrease in glycemic levels, insulin resistance index, lipids, C-reactive protein (CRP) levels, and creatinine levels in patients receiving RYBG. It was correlated with greater decreases in BMI, fasting insulin, insulin resistance index, and C-reactive protein levels in patients receiving OAGB. CONCLUSION: Diabetic patients receiving OAGB and RYGB had lower glucose and cholesterol levels compared with SG independent of weight loss. Our results suggest diabetic patients with cardiovascular risk factors such as hypercholesterolemia to receive bypass surgery.

Genome-Wide Association Studies for Albuminuria of Nondiabetic Taiwanese Population.

INTRODUCTION: Chronic kidney disease, which is defined by a reduced estimated glomerular filtration rate and albuminuria, imposes a large health burden worldwide. Ethnicity-specific associations are frequently observed in genome-wide association studies (GWAS). This study conducts a GWAS of albuminuria in the nondiabetic population of Taiwan. METHODS: Nondiabetic individuals aged 30-70 years without a history of cancer were enrolled from the Taiwan Biobank. A total of 6,768 subjects were subjected to a spot urine examination. After quality control using PLINK and imputation using SHAPEIT and IMPUTE2, a total of 3,638,350 single-nucleotide polymorphisms (SNPs) remained for testing. SNPs with a minor allele frequency of less than 0.1% were excluded. Linear regression was used to determine the relationship between SNPs and log urine albumin-to-creatinine ratio. RESULTS: Six suggestive loci are identified in or near the FCRL3 (p = 2.56 × 10-6), TMEM161 (p = 4.43 × 10-6), EFCAB1 (p = 2.03 × 10-6), ELMOD1 (p = 2.97 × 10-6), RYR3 (p = 1.34 × 10-6), and PIEZO2 (p = 2.19 × 10-7). Genetic variants in the FCRL3 gene that encode a secretory IgA receptor are found to be associated with IgA nephropathy, which can manifest as proteinuria. The PIEZO2 gene encodes a sensor for mechanical forces in mesangial cells and renin-producing cells. Five SNPs with a p-value between 5 × 10-6 and 5 × 10-5 are also identified in five genes that may have a biological role in the development of albuminuria. CONCLUSION: Five new loci and one known suggestive locus for albuminuria are identified in the nondiabetic Taiwanese population.

Survey Outcome on Immunogenicity Risk Assessment Tools for Biotherapeutics: an Insight into Consensus on Methods, Application, and Utility in Drug Development.

A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.

Prediabetes and risk of active tuberculosis: a cohort study from Northern Taiwan.

BACKGROUND: Diabetes mellitus (DM) is a well-established risk factor for active tuberculosis (TB) infection. Despite the worldwide rapid increase in the prevalence of prediabetes, its impact on the risk of active TB remains largely unknown. This study aimed to investigate the relationship between prediabetes and risk of active TB in a large cohort study. METHODS: A total of 119 352 participants were screened from a community-based health screening programme in Northern Taiwan. Diabetes mellitus and prediabetes were defined by baseline fasting plasma glucose (FPG) and prescription of anti-diabetic drugs. Incident cases of active TB were identified from the National Tuberculosis Registry. Kaplan-Meier curves and Cox regression analysis were employed to estimate the hazard ratios for prediabetes and DM compared with normoglycaemia. Spline regression was performed to investigate the dose-response relationship between FPG level and risk of TB disease. RESULTS: At baseline, 27 404 (22.96%) participants had prediabetes and 10 943 (9.17%) participants had DM. After an average follow-up of 7.2 years, 322 TB cases occurred. The adjusted hazard ratio of developing active TB disease was 0.73 [95% confidence interval (CI) 0.55-0.97] for prediabetic and 1.48 (95% CI 1.11-1.98) for diabetic participants compared with normoglycaemic individuals. Spline regression revealed a U-shaped association between FPG level and risk of active TB disease, with the lowest risk at FPG around110 mg/dl. Sensitivity analyses were conducted to exclude factors such as potential confounders (including body mass index), misclassification of glycaemic level, and selection bias, and results showed that those factors could not explain the lower risk of active TB. CONCLUSIONS: Prediabetes was associated with a 27% reduced risk of active TB disease compared with normoglycaemia. The biological mechanism of this inverse association and its implication for global nutrition transition and TB control should be further investigated.

The Relationship between Obesity-Related Factors and Graves' Orbitopathy: A Pilot Study.

Background and Objectives: The aim of this study was to investigate the relationships between obesity-related factors including body mass index (BMI), diabetes or prediabetes, hyperlipidemia, fasting plasma glucose, fasting plasma insulin, homeostasis model assessment-estimated insulin resistance (HOMA-IR), highly sensitive C-reactive protein (hs-CRP) and Graves' orbitopathy (GO). Materials and Methods: Eighty-four patients with Graves' disease (GD) (42 without GO and 42 with GO) were enrolled in this cross-sectional cohort study. Gender, age, GD treatment history, height, body weight, waist circumference, smoking status, co-morbidities, levels of free thyroxin, thyroid-stimulating hormone, thyroid-stimulating hormone receptor (TSHR) antibodies, fasting plasma glucose and insulin, and hs-CRP were recorded. The eye condition was evaluated using the consensus statement of the European Group of Graves' Orbitopathy (EUGOGO) and the NOSPECS classification. Results: In this study, multivariate regression analysis showed that BMI, fasting plasma insulin, and HOMA-IR were associated with the presence of GO after adjusting the age, gender, smoking, TSHR antibodies, and steroid usage (adjusted odd's ratio (aOR) 1.182, 95% confidence interval (95% CI), 1.003-1.393, p = 0.046; aOR 1.165, 95% CI, 1.001-1.355, p = 0.048; and aOR 1.985, 95% CI, 1.046-3.764, p = 0.036, respectively). In addition, BMI, fasting plasma glucose, fasting plasma insulin, HOMA-IR, and hs-CRP levels were positively correlated with the severity of GO. Conclusions: The findings of this study suggest that obesity-related factors, especially fasting plasma insulin and HOMA-IR, are related to GO. Our study highlighted the importance of obesity-related factors in GO. Obesity-related factors may cause the development of GO or occur simultaneously with GO.

A genome-wide association study for melatonin secretion.

Melatonin exerts a wide range of effects among various tissues and organs. However, there is currently no study to investigate the genetic determinants of melatonin secretion. Here, we conducted a genome-wide association study (GWAS) for melatonin secretion using morning urine 6-hydroxymelatonin sulfate-to-creatinine ratio (UMCR). We initially enrolled 5000 participants from Taiwan Biobank in this study. After excluding individuals that did not have their urine collected in the morning, those who had history of neurological or psychiatric disorder, and those who failed to pass quality control, association of single nucleotide polymorphisms with log-transformed UMCR adjusted for age, sex and principal components of ancestry were analyzed. A second model additionally adjusted for estimated glomerular filtration rate (eGFR). A total of 2373 participants underwent the genome-wide analysis. Five candidate loci associated with log UMCR (P value ranging from 6.83 × 10-7 to 3.44 × 10-6) encompassing ZFHX3, GALNT15, GALNT13, LDLRAD3 and intergenic between SEPP1 and FLJ32255 were identified. Similar results were yielded with further adjustment for eGFR. Interestingly, the identified genes are associated with circadian behavior, neuronal differentiation, motor disorders, anxiety, and neurodegenerative diseases. We conducted the first GWAS for melatonin secretion and identified five candidate genetic loci associated with melatonin level. Replication and functional studies are needed in the future.

Characterization of the robust humoral immune response to GSK2618960, a humanized anti-IL-7 receptor monoclonal antibody, observed in healthy subjects in a Phase 1 study.

Interleukin-7 (IL-7) signaling modulates T cell activity and is implicated in numerous autoimmune diseases. An anti-IL-7 receptor monoclonal antibody (GSK2618960) biotherapeutic was evaluated in healthy subjects for safety, pharmacokinetics (PK), pharmacodynamics (PD) and immunogenicity in a single-dose escalation phase I study. We found that antibodies against GSK2618960 (i.e., anti-drug antibodies or ADA) developed in 83% and 100% of GSK2618960-treated subjects in the 0.6 and 2.0 mg/kg dose cohorts, respectively. Of the ADA positive subjects, 64% (7 of 11) had detectable neutralizing activity. Further investigation revealed the presence of GSK2618960-specific memory B cells, indicating the development of immunological memory for the ADAs. Ex vivo stimulation of peripheral blood mononuclear cell (PBMC) samples demonstrated a relatively strong CD4+ T cell proliferation response to GSK2618960 as compared to the control anti-RSV antibody (which is known to have only low immunogenic potential), confirming the high immunogenic potential of GSK2618960. Furthermore, GSK2618960 was found to bind in vitro monocyte-derived dendritic cells (DCs). GSK2618960 treatment of PBMCs increased the proportion of DC cells showing an increase in expression of CD83, CD86 and CD209, which indicated enhanced DC differentiation and activation relative to the isotype control anti-ß amyloid antibody. Collectively, the evidence supports that the high incidence of observed clinical immunogenicity was likely related to the receptor-mediated activity by GSK2618960.

Detection of Memory B Activity Against a Therapeutic Protein in Treatment-Naïve Subjects.

Bridging immunoassays commonly used to detect and characterize immunogenicity during biologic development do not provide direct information on the presence or development of a memory anti-drug antibody (ADA) response. In this study, a B cell ELISPOT assay method was used to evaluate pre-existing ADA for anti-TNFR1 domain antibody, GSK1995057, an experimental biologic in treatment naive subjects. This assay utilized a 7-day activation of PBMCs by a combination of GSK1995057 (antigen) and polyclonal stimulator followed by GSK1995057-specific ELISPOT for the enumeration of memory B cells that have differentiated into antibody secreting cells (ASC) in vitro. We demonstrated that GSK1995057-specific ASC were detectable in treatment-naïve subjects with pre-existing ADA; the frequency of drug-specific ASC was low and ranged from 1 to 10 spot forming units (SFU) per million cells. Interestingly, the frequency of drug-specific ASC correlated with the ADA level measured using an in vitro ADA assay. We further confirmed that the ASC originated from CD27+ memory B cells, not from CD27--naïve B cells. Our data demonstrated the utility of the B cell ELISPOT method in therapeutic protein immunogenicity evaluation, providing a novel way to confirm and characterize the cell population producing pre-existing ADA. This novel application of a B cell ELISPOT assay informs and characterizes immune memory activity regarding incidence and magnitude associated with a pre-existing ADA response.

Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive blocking antibody.

Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex. We demonstrated that the elevated drug-bound IL-5 produced false-positive response in the in vitro ADA assay, in which drug-bound IL-5 dissociated and then bridged mepolizumab conjugates to yield positive signal. To eliminate the IL-5 interference, we compared two strategies: a solid-phase immunodepletion of IL-5 and an in-solution IL-5 immunocompetition. We identified the best competitive antibody for each purpose. We found both methods demonstrated similar effectiveness in reducing the false positive signal in IL-5 spiked samples; however, the in-solution immunocompetition for IL-5 had fewer false positives in study samples. Additionally, the in-solution immunocompetition method was experimentally simpler to execute. We modified the ADA assay by adding a pre-treatment step with a mepolizumab competitive anti- IL-5 antibody. Using this new method, we retested clinical samples from two phase II studies (MEA112997 and MEA114092). The confirmed ADA positive incidence was reduced from 29% and 61% to 1% and 8% with the modified in-solution immune inhibition method. Target interference is a fairly common problem facing immunogenicity testing, and target-induced false positive cannot be distinguished from true ADA response by the commonly used drug competitive confirmation assay. The approach and method used here for resolving target interference in ADA detection will be useful for differentiating between a true ADA response and target induced false positive as well as similar challenges in other programs.

Exploitation of the size-exclusion effect of reversed-phase high performance liquid chromatography for the direct analysis of diethylene triamine pentaacetic acid in therapeutic monoclonal antibody formulations.

Monoclonal antibodies (mAb) are being widely studied for the treatment of cancers and other diseases. The mAb is typically in a solution formulation and administered as an intravenous infusion. Ready-to-use solutions are favored for their clinical convenience but they can potentially suffer from a shorter shelf life due to accelerated rates of some forms of degradation such as oxidation, relative to lyophilized formulations. To improve stability, the chelating agent diethylene triamine pentaacetic acid (DTPA) is often used at very low concentrations in biologics formulations to prevent oxidation induced by metal ions. Because of its low concentration and susceptibility to changes in concentration during stability study or processing, the measurement of DTPA levels during formulation and process development is critical. In response to this need we developed a platform reversed-phase HPLC method that allows for the rapid and direct determination of DPTA concentrations which does not require the prior removal of mAbs in formulation samples. The method exploits the "size exclusion effect" of C18 columns with narrow pore sizes (90-120Å) to elute large mAb at the void volume, enabling direct injections of mAb samples for quantitation of DTPA. The method was found to be suitable for the analysis of DTPA in the range of 2-20µg/mL across multiple drug formulations containing different therapeutic mAb and antibody drug conjugates. The method was successfully validated for specificity, precision, accuracy, linearity, and robustness.

Development of an enzymatic assay for the detection of neutralizing antibodies against therapeutic angiotensin-converting enzyme 2 (ACE2).

Therapeutic proteins have the potential to elicit immune responses in animals and humans (Mire-Sluis et al., 2004; Yu et al., 2006; Shankar et al., 2008). Contributors to the response could include product related factors such as chemical modifications, impurities that co-purify with product, contaminants, formulation, aggregates, and clinical factors such as dose concentration, dosing frequency, route of drug administration, rate of administration, patient underlying disease, concomitant medication, and genetic status among others (Patten and Schellekens, 2003). Further, an immune response triggered by a therapeutic enzyme may neutralize the endogenous counterpart resulting in a decrease or depletion of the therapeutic and endogenous enzymes imposing safety concerns for patients. Therefore, monitoring of anti-drug antibody (ADA) and neutralizing antibody (NAb) responses to both the recombinant therapeutic enzyme and endogenous enzyme is important during early development and subsequent clinical studies. Testing considerations for NAb detection against therapeutic enzymes have been published mostly for lysosomal storage diseases (Wang et al., 2008). NAb cross-reactivity to the endogenous counterpart has also been characterized (Sominanda et al., 2010). Here, we describe an enzymatic NAb assay which detects neutralizing antibodies to both recombinant and endogenous angiotensin-converting enzyme 2 (ACE2). NAb assay sensitivity was optimized by selecting the assay incubation time as 20 min with an enzyme concentration of 0.5 µg/mL. Four anti-ACE2 antibodies out of a commercial panel of 18 were found to have neutralizing capabilities based upon their ability to abrogate ACE2 enzymatic activity. We demonstrated assay specificity by small peptide inhibitors specific for ACE or ACE2. DX600, an ACE2 specific inhibitor did not cross-react with ACE. Conversely, captopril, an inhibitor of ACE did not inhibit ACE2. The assay specificity for ACE2 neutralizing antibodies was further demonstrated by the lack of reactivity of two species control antibodies and 14 anti-ACE2 antibodies. Moreover, we demonstrated assay specificity to human endogenous ACE2 from human epithelial cells. Three human cell lines (Calu-3, Caco-2, Huh-7) were evaluated for the cell surface expression of ACE2 by flow cytometry and Western blot. Subsequently, whole cell lysates, cell culture supernatant, and live cells were evaluated in the assay. Results demonstrated that Calu-3 had elevated levels of ACE2 compared to Caco-2 or Huh-7. Calu-3 also demonstrated elevated ACE2 enzymatic activity in all three sources and could be inhibited by the ACE2 specific inhibitor DX600 as well as the neutralizing antibodies for the recombinant ACE2. Thus, we describe here a method to detect NAb against a therapeutic enzyme and assess NAb cross-reactivity to the native endogenous enzyme. The approach of method development described here could be applied for the assessment of NAb responses to other enzymatic therapeutics.

Development and validation of a cell-based SEAP reporter assay for the detection of neutralizing antibodies against an anti-IL-13 therapeutic antibody.

A cell-based bioassay capable of detecting neutralizing antibodies (NAb) specific to a therapeutic anti-IL-13 monoclonal antibody was developed, validated and used to analyze normal human and asthma serum samples. At the time of this study, a neutralizing assay was unavailable for anti-IL-13 antibody therapeutics with sufficient rigor for validation. Thus, we describe here a method and considerations for validation. The assay used IL-13 responsive HEK293 cells transfected with a secreted embryonic alkaline phosphatase (SEAP) reporter gene. Cells were plated at 5.4×10(4) per assay well due to 90% confluence on the subsequent day. Optimal IL-13 and anti-IL-13 concentrations were determined to be 600 pg/mL and 900 ng/mL respectively. We demonstrated the assay's cut point, sensitivity, specificity/cross reactivity, selectivity/matrix interference, and precision. Also, we demonstrated how the drug inhibitory concentration (IC(50), IC(75), and IC(90)) can affect sensitivity and dynamic range/assay window. We characterized the differences in assay response between serum samples of normal population and asthma population. Asthma samples demonstrated an elevated OD ratio in average compared to normal samples. Thus, separate cut points were needed and calculated to be 1.78 and 2.43 for normal and asthma serum, respectively. The assay sensitivity was 670 ng/mL with the positive control (affinity purified rabbit anti-drug polyclonal antibodies). Potential false positives resulting from endogenous serum cytokines including IL-13, IL-4, and Interferon alpha (INF-α) were evaluated and the results indicated that the interfering concentrations for these cytokines are much higher than the respective physiological concentrations. Based on these data, the risk of false positive by endogenous cytokines was considered to be low. In addition, irrelevant anti-drug positive control antibodies were evaluated for assay specificity and did not demonstrate neutralizing capability. Further, no matrix interference in the intended patient population was found when using a final assay serum concentration of 16.7%. The validated assay had acceptable intra- and inter- assay precision in that all %CVs were ≤25%. Overall, this assay successfully proceeded through validation and was used to determine NAb responses within serum samples.