Biologic drugs (therapeutic proteins or peptides) have become one of the most important therapeutic modalities over the past few decades. Drug-induced immunogenicity is a significant concern as it may affect safety, tolerability, and efficacy. With more sensitive and drug-tolerant screening assays in use today, reliable estimation of anti-drug-antibody (ADA) titer has become more important for understanding clinically relevant ADA levels. Titer is commonly defined as the dilution factor resulting in an assay signal equal to a pre-specified cut point factor. Given its influence on the resulting titer precision, the choice of a titer cut point factor warrants careful consideration. In this paper, we discuss the theoretical dilution model, investigate how titer variability depends on the cut point factor and propose a standardized cut point factor to increase precision of titer estimates. Additionally, we demonstrate that non-linear regression-based titer estimation provides both improved precision and implementation efficiency relative to commonly used estimation approaches.

We previously demonstrated that angiopoietin-like protein 8 (ANGPTL8) forms ANGPTL3/8 and ANGPTL4/8 complexes that increase with feeding to direct fatty acids (FA) toward adipose tissue through differential modulation of lipoprotein lipase (LPL) activity. Each complex correlated inversely with high density lipoprotein cholesterol (HDL) in control subjects. We thus investigated ANGPTL3/8 and ANGPTL4/8 levels in type 2 diabetes patients, who can present with decreased HDL. While ANGPTL3/8 levels in type 2 diabetes patients were similar to those previously observed in normal controls, ANGPTL4/8 levels were roughly twice as high as those in control subjects. Concentrations of ANGPTL3/8 and ANGPTL4/8 in type 2 diabetes patients were inversely correlated with HDL, with the correlation being significant for ANGPTL4/8. We therefore measured the ability of the various ANGPTL proteins and complexes to inhibit endothelial lipase (EL), the enzyme which hydrolyzes phospholipids (PL) in HDL. While confirming ANGPTL3 as an EL inhibitor, we found that ANGPTL4 was a more potent EL inhibitor than ANGPTL3. Interestingly, we observed that while ANGPTL3/8 had increased EL-inhibitory activity compared to ANGPTL3 alone, ANGPTL4/8 exhibited decreased potency in inhibiting EL compared to ANGPTL4 alone. Together, these results show for the first time that ANGPTL4 is a more potent EL inhibitor than ANGPTL3 and suggest a possible reason for why ANGPTL4/8 levels are correlated inversely with HDL.

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.

Triglyceride (TG) molecules represent the major storage form of fatty acids, and TG metabolism is essential to human health. However, the mechanistic details surrounding TG metabolism are complex and incompletely elucidated. Although it is known that angiopoietin-like protein 8 (ANGPTL8) increases TGs through an ANGPTL3/8 complex that inhibits LPL, the mechanism governing ApoA5, which lowers TGs, has remained elusive. Current hypotheses for how ApoA5 acts include direct stimulation of LPL, facilitation of TG-containing particle uptake, and regulation of hepatic TG secretion. Using immunoprecipitation-MS and Western blotting, biolayer interferometry, functional LPL enzymatic assays, and kinetic analyses of LPL activity, we show that ApoA5 associates with ANGPTL3/8 in human serum and most likely decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition. We also demonstrate that ApoA5 has no direct effect on LPL, nor does it suppress the LPL-inhibitory activities of ANGPTL3, ANGPTL4, or ANGPTL4/8. Importantly, ApoA5 suppression of ANGPTL3/8-mediated LPL inhibition occurred at a molar ratio consistent with the circulating concentrations of ApoA5 and ANGPTL3/8. Because liver X receptor (LXR) agonists decrease ApoA5 expression and cause hypertriglyceridemia, we investigated the effect of the prototypical LXR agonist T0901317 on human primary hepatocytes. We observed that T0901317 modestly stimulated hepatocyte ApoA5 release, but markedly stimulated ANGPTL3/8 secretion. Interestingly, the addition of insulin to T0901317 attenuated ApoA5 secretion, but further increased ANGPTL3/8 secretion. Together, these results reveal a novel intersection of ApoA5 and ANGPTL3/8 in the regulation of TG metabolism and provide a possible explanation for LXR agonist-induced hypertriglyceridemia.

We previously demonstrated that angiopoietin-like 8 (ANGPTL8) forms a localized complex with ANGPTL4 to reduce its lipoprotein lipase (LPL)-inhibitory activity and enable increased postprandial uptake of fatty acids (FA) into adipose tissue. Because prolonged cold exposure may increase adipose tissue FA uptake and decrease circulating triglycerides (TG) by reducing ANGPTL4 expression and inducing ANGPTL8 expression (and thus ANGPTL4/8 expression), we investigated the effect of temperature on ANGPTL4 and ANGPTL4/8 LPL-inhibitory activities in vitro. As the ANGPTL4(E40K) mutation results in decreased TG, we also characterized ANGPTL4(E40K) and ANGPTL4(E40K)/8 complex LPL-inhibitory activities. Interestingly, while ANGPTL3, ANGPTL3/8, and ANGPTL4 showed similar LPL inhibition at 37 °C and 22 °C, the already reduced LPL-inhibitory activity of ANGPTL4/8 at 37 °C was even more decreased at 22 °C. At 37 °C, ANGPTL4(E40K) manifested decreased LPL-inhibitory activity compared to ANGPTL4/8, while ANGPTL4(E40K)/8 had even further reduced potency. Remarkably, ANGPTL4/8, ANGPTL4(E40K), and ANGPTL4(E40K)/8 were each actually capable of stimulating LPL activity at 22 °C. Together, these results indicate that ANGPTL4/8 stimulation of LPL activity at low temperatures may represent an additional mechanism for further increasing adipose tissue FA uptake during cold exposure, beyond that already occurring due to decreased ANGPTL4 expression and increased ANGPTL8 expression. In addition, because ANGPTL4(E40K) has decreased LPL-inhibitory activity compared to ANGPTL4/8, our findings also suggest why ANGPTL4(E40K) carriers have decreased circulating TG levels.

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.

Angiopoietin-like protein (ANGPTL)8 has been implicated in metabolic syndrome and reported to regulate adipose FA uptake through unknown mechanisms. Here, we studied how complex formation of ANGPTL8 with ANGPTL3 or ANGPTL4 varies with feeding to regulate LPL. In human serum, ANGPTL3/8 and ANGPTL4/8 complexes both increased postprandially, correlated negatively with HDL, and correlated positively with all other metabolic syndrome markers. ANGPTL3/8 also correlated positively with LDL-C and blocked LPL-facilitated hepatocyte VLDL-C uptake. LPL-inhibitory activity of ANGPTL3/8 was >100-fold more potent than that of ANGPTL3, and LPL-inhibitory activity of ANGPTL4/8 was >100-fold less potent than that of ANGPTL4. Quantitative analyses of inhibitory activities and competition experiments among the complexes suggested a model in which localized ANGPTL4/8 blocks the LPL-inhibitory activity of both circulating ANGPTL3/8 and localized ANGPTL4, allowing lipid sequestration into fat rather than muscle during the fed state. Supporting this model, insulin increased ANGPTL3/8 secretion from hepatocytes and ANGPTL4/8 secretion from adipocytes. These results suggest that low ANGPTL8 levels during fasting enable ANGPTL4-mediated LPL inhibition in fat tissue to minimize adipose FA uptake. During feeding, increased ANGPTL8 increases ANGPTL3 inhibition of LPL in muscle via circulating ANGPTL3/8, while decreasing ANGPTL4 inhibition of LPL in adipose tissue through localized ANGPTL4/8, thereby increasing FA uptake into adipose tissue. Excessive caloric intake may shift this system toward the latter conditions, possibly predisposing to metabolic syndrome.

Safety, tolerability, and pharmacology profiles of LY3127760, an EP4 antagonist, were explored in healthy subjects in a subject/investigator-blind, parallel-group, multiple-ascending dose study. Cohorts consisted of 13 patients randomized to LY3127760, celecoxib (400 mg), or placebo (9:2:2 ratio) for 28 days. LY3127760 was well tolerated; the most commonly observed adverse events were gastrointestinal, similar to celecoxib. LY3127760 increased release of ex vivo tumor necrosis factor alpha after lipopolysaccharide/prostaglandin E2 stimulation when compared with placebo, suggesting a dose-dependent blockade of the EP4 receptor. Compared with placebo, 24-h urinary excretion of prostaglandin E metabolite was modestly increased; prostacyclin metabolite was inhibited; and thromboxane A2 metabolite was unchanged. Effects on sodium and potassium excretion were similar to those of celecoxib. We conclude that LY3127760 demonstrated similar effects on prostacyclin synthesis and renal sodium retention as celecoxib. These data support exploration of LY3127760 at daily doses of 60 mg to 600 mg in phase II trials. This trial's registration number: NCT01968070.

Background: Increased emphasis on the development of biologics has placed a significant focus on anti-drug antibody (ADA) detection. To address this need, several immunoassay formats have been described for use in characterizing potential immune responses. Two commonly utilized methods include the affinity capture elution (ACE) and bridging formats. While these approaches have been effective in supporting many clinical initiatives, both possess potential disadvantages. Here, we compare these standard methods to a novel format that addresses these noted drawbacks.Results: A novel assay format has been designed to incorporate the benefits of the ACE and bridging methods while overcoming the disadvantages incurred with each approach. The described ACE-Bridge format exhibits excellent sensitivity and precision while providing superior drug tolerance when compared to bridging formats. Further, this assay format is not susceptible to the endogenous target interference that can be an issue in the ACE format.Conclusions: The ACE-Bridge format provides an often superior option as a screening method to monitor patient ADA responses. This method is unique in its ability to measure ADA in the presence of high circulating endogenous target concentrations (>100 ng/mL) while demonstrating very high drug tolerance. (C) 2016 Elsevier B.V. All rights reserved.

AIMImmunogenicity testing of biotherapeutic drugs is a regulatory requirement. Herein, we describe a drug-tolerant assay for detecting neutralizing antibodies against a therapeutic antibody.RESULTSExcess target of the therapeutic antibody was incorporated into the detection step of an affinity capture elution assay. Signal generated from binding of antidrug antibody (ADA) to the therapeutic antibody was compared with signal from binding of ADA to the therapeutic antibody preincubated with its target. The results demonstrated that the target blocked binding of the therapeutic antibody to neutralizing monkey ADA and to two anti-idiotypic antibodies.CONCLUSIONThis highly drug-tolerant novel approach enables the detection of neutralizing antibodies and allows for one basic assay format to achieve complete characterization of ADA responses.

The mechanisms involved in chronic pain and inflammation have come under intense scrutiny in the last two decades. With non-steroidal antiinflammatory drugs (NSAIDs) providing suboptimal relief for inflammatory pain, the targeting of other pro-inflammatory pathways is being investigated for therapeutic application. The purinergic receptor P2X7 is thought to regulate the activation of caspase-1, which subsequently cleaves prointerleukin-1β (IL-1β), thereby causing the secretion of mature IL-1β. In this study, we utilized an ex vivo stimulation of human peripheral blood with variable duration lipopolysaccharide (LPS) priming and examined the effects of ATP, P2X7 antagonists, and NSAIDS on IL-1β and caspase-1. We observed that following LPS priming, the P2X7 agonist and ATP analogue BzATP markedly stimulated IL-1β secretion. Interestingly, BzATP also stimulated the secretion of caspase-1 itself in a dose and time-dependent manner that paralleled the secretion of IL-1β, which was also dependent upon LPS priming. The secretion of both IL-1β and caspase-1 was almost completely blocked by the P2X7 antagonists PPADS and AZ11645373. Moreover, secretion of both IL-1β and caspase-1 was inhibited by AZ11645373 in a parallel, dosedependent manner. In contrast, NSAIDS were unable to inhibit secretion of either IL-1β or caspase-1. These results suggest that both ATPinduced IL-1β secretion and caspase-1 secretion are mediated almost entirely through the P2X7 receptor. These data also further indicate that by inhibiting IL-1β and caspase-1 secretion, P2X7 antagonism may represent a novel mechanism to inhibit pain and inflammation that is not achievable with currently available NSAIDS.

Unraveling the role of proprotein convertase subtilisin kexin type 9 (PCSK9) as a key posttranscriptional regulator of the LDL receptor (LDLR) has shed light on its attractiveness as a target for LDL cholesterol (LDL-C) lowering. PCSK9 is a serine protease that is secreted by the liver upon post translational self-cleavage of its pro-domain. Once in circulation, the protein binds the hepatic LDLR, and upon internalization, the receptor is sequestered for lysosomal destruction. Gain-of-function mutations for PCSK9 induce familial hypercholesterolemia, whereas loss-offunction mutations display a broad range of mutation dependent LDL-C lowering. Initial mutation and knockout analyses established the correlation between PCSK9 and circulating LDL-C. Ensuing RT-PCR studies demonstrated statin induced up-regulation of PCSK9 mRNA expression. Subsequently, the development of novel monoclonal PCSK9 antibodies facilitated sensitive and robust ligand-binding assays that have produced concrete conclusions linking PCSK9 to LDL-C regulation. These methods have demonstrated the impacts of current lipid lowering therapies on levels of circulating human PCSK9; furthermore, they will be a key component in addressing the pharmacodynamic responses of first generation PCSK9 lowering therapeutics. Here, we summarize and assess the methodologies for PCSK9 measurement in the context of a review of events that have led to our present knowledge about this intriguing target. Moreover, we address conflicting data and discuss ongoing bioanalytical challenges in understanding the true circulating levels of PCSK9. Finally, we discuss the pharmacological impact of PCSK9 measurement as a screening tool and a biomarker for future LDL-C lowering therapeutics.