INTRODUCTION:This analysis of the phase 3 TRAILBLAZER-ALZ 2 trial examined whether plasma tau protein phosphorylated at threonine 217 (p-tau217) level can reliably monitor treatment-related amyloid clearance (TRAC) after donanemab treatment in early symptomatic Alzheimer's disease (AD). METHODS:Amyloid positron emission tomography (PET) and plasma p-tau217 levels were assessed at baseline and longitudinally during study treatment. The diagnostic performance of p-tau217 in detecting post-treatment TRAC (< 24.1 Centiloids [CL]) was evaluated by receiver operating characteristic (ROC) analysis. RESULTS:Plasma p-tau217 showed suboptimal performance in detecting TRAC by PET in donanemab-treated participants (N = 830), with an area under the ROC curve of 0.61 at 52 weeks. DISCUSSION:Despite reductions in plasma p-tau217 with donanemab treatment, this biomarker cannot currently be used to accurately detect TRAC by PET (< 24.1 CL) in individuals with early symptomatic AD.

OBJECTIVE:Clinically relevant Alzheimer's disease co-pathology is common in Lewy body disorders. Plasma P-tau217 is a sensitive biomarker for amyloid and tau pathology in Alzheimer's disease. The objective was to determine if plasma P-tau217 associates with Alzheimer's disease co-pathology and cognition in Lewy body disorders. METHODS:Participants had (1) a clinicopathological diagnosis of Parkinson's disease or dementia with Lewy bodies in the pathology series, or (2) a clinical diagnosis of Parkinson's disease in the clinical series and were followed as part of the longitudinal, observational cohort study at the University of Pennsylvania between 2007 and 2024. Plasma concentration of P-tau217 was measured in previously collected samples. RESULTS:Neuropathology cases included 46 Parkinson's disease and 10 dementia with Lewy bodies, and clinical cases included 173 Parkinson's disease, and 64 controls. P-tau217 was greater in cases with (median, 0.3 [interquartile range (IQR), 0.2-0.4]) versus without (median, 0.1 [IQR, 0.1-0.2]) Alzheimer's disease co-pathology (p < 0.01) and discriminates Lewy body disorders participants with Alzheimer's disease co-pathology (area under curve, 0.84). Parkinson's disease participants with incident cognitive impairment had greater increases in serial P-tau217 than those who remained cognitively stable (group × time interaction β = -0.010, p = 0.0027). Higher baseline P-tau217 concentrations associated with longitudinal change in dementia rating scale (group × time interaction β = -4.947, p = 0.0166) and higher risk for cognitive progression (hazard ratio = 1.597, p = 0.003). INTERPRETATION:Plasma P-tau217 detects Alzheimer's disease co-pathology in Lewy body disorders and predicts cognitive decline. Future studies will evaluate associations between plasma P-tau217 and imaging and clinical outcomes, in consideration for use of amyloid-targeting therapies in Lewy body disorders. ANN NEUROL 2026;99:1428-1437.

Formation of anti-drug antibodies (ADA) and, in particular, neutralizing antibodies (NAb), is a major risk to the development of biotherapeutics. Cell-based assays, which can reflect the mechanistic interactions among the drug, target, and NAb, are often most suitable for the detection of NAb. We report the development and validation of cell-based platform assays for a class of incretin molecules. An affinity capture elution step was employed to improve drug tolerance of a cell-based cyclic adenosine monophosphate (cAMP) assay. Assay conditions and procedures, including acid elution, cell density, and TAG labeled cAMP (cAMP-TAG) incubation time and concentration, were thoroughly optimized. Delta percent (Δ

INRODUCTION:Plasma phosphorylated tau217 (p-tau217) is a biomarker for the detection of amyloid pathology. We present performance characteristics of the Lilly SP-X P-tau217 assay in a clinical validation cohort, as a whole and divided into subpopulations from traditionally underrepresented groups. METHODS:We measured p-tau217 levels in plasma samples from participants with mild cognitive impairment. Assay performance in predicting positivity for amyloid beta (Aβ) positron emission tomography was examined using two numerical cutoffs. RESULTS:Two p-tau217 cutoffs were determined with an upper-level group with 95% positive predictive value for Aβ positivity and a lower-level group with 84% negative predictive value for Aβ negativity, with 91% sensitivity and 90% specificity. The remaining indeterminate group represented 18% of participant samples. Similar performance was observed across validation subgroups. Discussion:The validation data support the potential clinical utility of the SP-X p-tau217 assay in multiple subpopulations to aid in Alzheimer's disease diagnosis. Highlights:The Lilly SP-X p-tau217 assay showed strong concordance with amyloid PET in total cohort and underrepresented groups.Assay performance is in line with guidance from the Global CEOi on AD Working Group.Data support the clinical utility of the assay in multiple cohorts to aid in AD diagnosis.

Background: Blood-based biomarkers, especially P-tau217, have been gaining interest as diagnostic tools to measure Alzheimer disease (AD) pathology. Methods: We developed a plasma P-tau217 chemiluminescent immunoassay using 4G10E2 and IBA493 as antibodies, a synthetic tau peptide as calibrator, and the Quanterix SP-X imager. Analytical validation performed in a College of American Pathologists-accredited CLIA laboratory involved multiple kit lots, operators, timepoints, and imagers. Florbetapir positron emission tomography was used to quantify amyloid for clinical validation. Results: Precision across 80 runs was <= 20% CV using 23 patient-derived samples ranging from 0.09 U/mL to 3.35 U/mL. No significant lot-to-lot differences were observed. There was no interference from purified tau (2N4R) or lipemia, but hemolysis greater than 2 + was not acceptable. Functional analytical sensitivity (lower limit of quantitation) was 0.08 U/mL. Linearity studies support the use of a standard 1:2 plasma dilution. Samples demonstrated stability at 7 freeze/thaw cycles, with room temperature and refrigerated stability established for up to 72 hours. The final analytical measurement range was 0.08 to 2.81 U/mL. The calibration curve maintained <= 20% CV for raw signal intensity and 80% to 120% relative error for back-fitted concentration using a log-log power regression. Initial clinical assessment using plasma samples from 1091 individuals screened in TRAILBLAZER-ALZ 2 demonstrated an area under the curve of 91.6% (95% CI 0.90-0.94) with brain amyloid as the comparator. Positive and negative predictive value was >90% and >85%, respectively. Conclusions: Through analytical validation, this assay demonstrated robust performance across multiple lots, operators, and instruments and could be used as a tool for diagnosing AD.

Context Antidrug antibodies (ADA) can potentially affect drug pharmacokinetics, safety, and efficacy.Objective This work aimed to evaluate treatment-emergent (TE) ADA in tirzepatide (TZP)-treated participants across 7 phase 3 trials and their potential effect on pharmacokinetics, efficacy, and safety.Methods ADA were assessed at baseline and throughout the study until end point, defined as week 40 (SURPASS-1, -2, and -5) or week 52 (SURPASS-3, -4, Japan-Mono, and Japan-Combo). Samples for ADA characterization were collected at SURPASS trial sites. Participants included ADA-evaluable TZP-treated patients with type 2 diabetes (N = 5025). Interventions included TZP 5, 10, or 15 mg. ADA were detected and characterized for their ability to cross-react with native glucose-dependent insulinotropic polypeptide (nGIP) and glucagon-like peptide-1 (nGLP-1), neutralize tirzepatide activity on GIP and GLP-1 receptors, and neutralize nGIP and nGLP-1.Results TE ADA developed in 51.1% of tirzepatide-treated patients. Proportions were similar across dose groups. Maximum ADA titers ranged from 1:20 to 1: 81 920 among TE ADA+ patients. Neutralizing antibodies (NAb) against TZP activity on GIP and GLP-1 receptors were observed in 1.9% and 2.1% of patients, respectively. Less than 1.0% of patients had cross-reactive NAb against nGIP or nGLP-1. TE ADA status, ADA titer, and NAb status had no effect on the pharmacokinetics or efficacy of TZP. More TE ADA+ patients experienced hypersensitivity reactions or injection site reactions than TE ADA- patients. The majority of hypersensitivity and injection site reactions were nonserious and nonsevere, and most events occurred and/or resolved irrespective of TE ADA status or titer.Conclusion Immunogenicity did not affect TZP pharmacokinetics or efficacy. The majority of hypersensitivity or injection site reactions experienced by TE ADA+ patients were mild to moderate in severity.

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.

Metal binding by members of the growth hormone (GH) family of hematopoietic cytokines has been a subject of considerable interest. However, beyond appreciation of its role in reversible packing of GH proteins in secretory granules, the molecular mechanisms of metal binding and granule formation remain poorly understood. Here, we investigate metal binding by a GH family member prolactin (PRL) using paramagnetic metal titration and chelation experiments. Cu2+-mediated paramagnetic relaxation enhancement measurements identified two partial metal-binding sites on the opposite faces of PRL composed of residues H30/H180 and E93/H97, respectively. Coordination of metal ions by these two sites causes formation of inter-molecular bridges between the PRL protomers and enables formation of reversible higher aggregates. These findings in vitro suggest a model for reversible packaging of PRL in secretory granules. The proposed mechanism of metal-promoted PRL aggregation lends insight and support to the previously suggested role of metal coordination in secretory granule formation by GH proteins.

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.

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.

Evolving immunogenicity assay performance expectations and a lack of harmonized anti-drug antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. Following debate at the American Association of Pharmaceutical Sciences National Biotechnology Conference, a group was formed to address these gaps. Over the last 3 years, 44 members from 29 organizations (including 5 members from Europe and 10 members from FDA) discussed gaps in understanding immunogenicity assay requirements and have developed harmonization tools for use by industry scientists to facilitate filings to health authorities. Herein, this team provides testing and reporting strategies and tools for the following assessments: (1) pre-study validation cut point; (2) in-study cut points, including procedures for applying cut points to mixed populations; (3) system suitability control criteria for in-study plate acceptance; (4) assay sensitivity, including the selection of an appropriate low positive control; (5) specificity, including drug and target tolerance; (6) sample stability that reflects sample storage and handling conditions; (7) assay selectivity to matrix components, including hemolytic, lipemic, and disease state matrices; (8) domain specificity for multi-domain therapeutics; (9) and minimum required dilution and extraction-based sample processing for titer reporting.

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.

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.

CXCL12 activates CXCR4 and is involved in embryogenesis, hematopoiesis, and angiogenesis. It has pathological roles in HIV-1, WHIM disease, cancer, and autoimmune diseases. An antagonist, AMD3100, is used for the release of CD34+ hematopoietic stem cells from the bone marrow for autologous transplantation for lymphoma or multiple myeloma patients. Adverse effects are tolerated due to its short-term treatment, but AMD3100 is cardiotoxic in clinical studies for HIV-1. In an effort to determine whether Saccharomyces cerevisiae expressing a functional human CXCR4 could be used as a platform for identifying a ligand from a library of less ∼1,000 compounds, a high-throughput screening was developed. We report that 2-carboxyphenyl phosphate (fosfosal) up-regulates CXCR4 activation only in the presence of CXCL12. This is the first identification of a compound that increases CXCR4 activity by any mechanism. We mapped the fosfosal binding site on CXCL12, described its mechanism of action, and studied its chemical components, salicylate and phosphate, to conclude that they synergize to achieve the functional effect.

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-naive, 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.

May 5, 2019April 9, 2019Free AccessImmunogenicity Assessment from Phase 3 Galcanezumab Trials in Patients with Episodic or Chronic Migraine (P1.10-017)James M. Martinez, Nada Hindiyeh, Greg Anglin, Kavita Kalidas, Michael E. Hodsdon, William Kielbasa, Brian A. Moser, Eric Pearlman, and Sandra GarcesAuthors Info & AffiliationsApril 9, 2019 issue92 (15_supplement)https://doi.org/10.1212/WNL.92.15_supplement.P1.10-017 Letters to the Editor

Background Safety findings from a Phase 2b study of galcanezumab, a humanized monoclonal antibody against calcitonin gene-related peptide, for prevention of migraine (NCT02163993) are reported here. Methods Patients aged 18–65 years with episodic migraine were evaluated in this multicenter, double-blind, randomized study. After randomization, 410 patients were administered 5, 50, 120 or 300 mg of galcanezumab or placebo subcutaneously once every 4 weeks for 12 weeks, followed by a post-treatment off-drug period lasting 12 weeks. Results Treatment-emergent adverse events (TEAEs) were primarily rated as mild to moderate. Serious adverse events reported in galcanezumab dose groups were appendicitis, Crohn’s disease, suicidal ideation, and congenital ankyloglossia in an infant of a paternal pregnancy; each of these were reported by one patient. Adverse events leading to discontinuation with galcanezumab treatment were abdominal pain, visual impairment, and upper limb fracture, each reported by one patient. Treatment-emergent injection-site reactions were reported significantly more frequently ( p = 0.013) with galcanezumab (13.9%) than with placebo (5.8%). Injection-site pain was the most common injection-site reaction (galcanezumab 11.4%; placebo 2.9%, p = 0.004). Upper respiratory tract infection (galcanezumab 10.0%; placebo 8.8%) and nasopharyngitis (galcanezumab 7.0%; placebo 2.2%) also occurred more frequently with galcanezumab treatment. Potential hypersensitivity events were reported at similar frequencies in galcanezumab (3.3%) and placebo (5.1%) groups. Incidence of treatment-emergent anti-drug antibodies in galcanezumab dose groups (4.6% of patients during treatment period) did not appear to have any meaningful effects on safety, the pharmacokinetics of galcanezumab, or its ability to bind to the target ligand. Conclusion The results from this 3-month Phase 2b study support the initiation of larger Phase 3 trials of longer duration.

PC2 (polycystin-2) forms a Ca(2+)-permeable channel in the cell membrane and its function is regulated by cytosolic Ca(2+) levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) lead to autosomal dominant polycystic kidney disease. The HPC2 Cterm protein contains a Ca(2+)-binding site responsible for channel gating and function. To provide the foundation for understanding how Ca(2+) regulates the channel through the HPC2 Cterm, we characterized Ca(2+) binding and its conformational and dynamic responses within the HPC2 Cterm. By examining hydrogen-deuterium (H-D) exchange profiles, we show that part of the coiled-coil domain in the HPC2 Cterm forms a stable helix bundle regardless of the presence of Ca(2+). The HPC2 L1EF construct contains the Ca(2+)-binding EF-hand and the N-terminal linker 1 region without the downstream coiled coil. We show that the linker stabilizes the Ca(2+)-bound conformation of the EF-hand, thus enhancing its Ca(2+)-binding affinity to the same level as the HPC2 Cterm. In comparison, the coiled coil is not required for the high-affinity binding. By comparing the conformational dynamics of the HPC2 Cterm and HPC2 L1EF with saturating Ca(2+), we show that the HPC2 Cterm and HPC2 L1EF share a similar increase in structural stability upon Ca(2+) binding. Nevertheless, they have different profiles of H-D exchange under non-saturating Ca(2+) conditions, implying their different conformational exchange between the Ca(2+)-bound and -unbound states. The present study, for the first time, provides a complete map of dynamic responses to Ca(2+)-binding within the full-length HPC2 Cterm. Our results suggest mechanisms for functional regulation of the PC2 channel and PC2's roles in the pathophysiology of polycystic kidney disease.

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.

Polycystin-2 (PC2) is a Ca2+-regulated Ca2+-channel from the transient receptor potential (TRP) family. Mutations in PC2 can cause autosomal dominant polycystic kidney disease (ADPKD). The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is crucial for channel assembly and function. We have combined biophysical and structural approaches to characterize the Ca2+-dependent molecular mechanism within the C-terminal tail that is involved in channel assembly and functional regulation. We have determined that HPC2 Cterm forms a trimer in solution with and without Ca2+-bound, even though TRP channels are generally tetrameric. We have definitively shown that there is only one Ca2+-binding site in HPC2 Cterm, located within its EF-hand domain. However, its Ca2+-binding affinity is greatly enhanced relative to its intrinsic binding affinity in the isolated EF-hand, possibly due to the positive cooperativity from the trimer interaction. We also employed sea urchin PC2 as a parallel model to study. The sea urchin C-terminal domain construct (SUPC2 Ccore) also trimerizes in solution independent of Ca2+-binding. In contrast, the SUPC2 Ccore contains two Ca2+-binding sites within its EF-hand domain, which exhibit cooperative Ca2+-binding due to internal stabilization. Consequently, trimerization does not further improve Ca2+-binding affinity in SUPC2 Ccore relative to the isolated EF-hand domain. Using both hydrogen-deuterium exchange mass spectroscopy and nuclear magnetic resonance, we have localized the Ca2+-binding sites in PC2 C-terminal tail and mapped the conformational changes induced by Ca2+-binding. We demonstrate that in addition to the direct, local stabilizing effects within the EF-hand, Ca2+-binding also causes conformational changes in the distal coiled-coil domain. This study provides a structural basis for regulation of the PC2 channel by its cytosolic C-terminal domain, with an improved understanding of the functional role of PC2 in regulating intracellular Ca2+ signaling.