Compared with intravenous formulations, subcutaneous (s.c.) formulations of therapeutic monoclonal antibodies may provide increased patient access and more convenient administration options, although ...historically high‐volume s.c. administration (> 10–15 mL) has been challenging. We report results from two phase I studies in healthy participants (GP29523 and GP40201) that evaluated s.c. crenezumab, an anti‐Aβ monoclonal antibody in development for individuals at risk for autosomal‐dominant Alzheimer’s disease. GP29523 assessed safety, tolerability, and pharmacokinetics (PK) in 68 participants (aged 50–80 years) who received single ascending doses (600–7,200 mg) of crenezumab or placebo (4–40 mL). GP40201 assessed safety, tolerability, and PK in 72 participants (aged 18–80 years) who received different combinations of dose (1,700–6,800 mg), infusion volume (10–40 mL), and flow rate (2–4 mL/minute), with/without recombinant human hyaluronidase (rHuPH20). There were no serious or dose‐limiting adverse events in either study. There were no meaningful differences in pain scores among reference placebo (4 mL), test placebo (4–40 mL), or crenezumab (600–7,200 mg) in GP29523, or across treatments with varying infusion volume, flow rate, dose, or rHuPH20 co‐administration or concentration in GP40201. Transient erythema was the most common infusion site reaction in both studies. In GP40201 at volumes of ≥ 20 mL, rHuPH20 co‐administration appeared to reduce infusion site swelling incidence, but, in some cases, was associated with larger areas of infusion site erythema. Crenezumab exhibited approximately dose‐proportional PK, and s.c. bioavailability was 66% and independent of dose or rHuPH20 co‐administration. High‐dose, high‐concentration, high‐volume s.c. crenezumab formulated with/without rHuPH20 was well‐tolerated in healthy participants, with an acceptable safety profile.
Antibody-drug conjugates (ADCs) are monoclonal antibodies with covalently bound cytotoxic drugs. They are designed to target tumor antigens selectively and offer the hope of cancer treatment without ...the debilitating side-effects of conventional therapies. The concept of ADCs is not new; however, development of these therapeutics is challenging and only recently are promising clinical data emerging. These challenges include ADC bioanalysis, such as quantifying in serum/plasma for PK studies and strategies for assessing immunogenicity. ADCs have complex molecular structures incorporating large- and small-molecule characteristics and require diverse analytical methods, including ligand-binding assays and MS-based methods. ADCs are typically mixtures with a range of drug-to-antibody ratios. Biotransformations in vivo can lead to additional changes in drug-to-antibody ratios resulting in dynamically changing mixtures. Thus, a standard calibration curve consisting of the reference standard may not be appropriate for quantification of analytes in vivo and represents a unique challenge. This paper will share our perspective on why ADC bioanalysis is so complex and describe the strategies and rationale that we have used for ADCs, with highlights of original data from a variety of nonclinical and clinical case studies. Our strategy has involved novel protein structural characterization tools to help understand ADC biotransformations in vivo and use of the analyte knowledge gained to guide the development of quantitative bioanalytical assays.
Abstract
Background
Staphylococcus aureus is a leading cause of bacteremia, yet there remains a significant knowledge gap in the identification of relevant biomarkers that predict clinical outcomes. ...Heterogeneity in the host response to invasive S. aureus infection suggests that specific biomarker signatures could be utilized to differentiate patients prone to severe disease, thereby facilitating earlier implementation of more aggressive therapies.
Methods
To further elucidate the inflammatory correlates of poor clinical outcomes in patients with S. aureus bacteremia, we evaluated the association between a panel of blood proteins at initial presentation of bacteremia and disease severity outcomes using 2 cohorts of patients with S. aureus bacteremia (n = 32 and n = 124).
Results
We identified 13 candidate proteins that were correlated with mortality and persistent bacteremia. Prognostic modeling identified interleukin (IL)-8 and CCL2 as the strongest individual predictors of mortality, with the combination of these biomarkers classifying fatal outcome with 89% sensitivity and 77% specificity (P < .0001). Baseline IL-17A levels were elevated in patients with persistent bacteremia (P < .0001), endovascular (P = .026) and metastatic tissue infections (P = .012).
Conclusions
These results demonstrate the potential utility of selected biomarkers to distinguish patients with the highest risk for treatment failure and bacteremia-related complications, providing a valuable tool for clinicians in the management of S. aureus bacteremia. Additionally, these biomarkers could identify patients with the greatest potential to benefit from novel therapies in clinical trials.
Biomarkers identify S. aureus bacteremia patients at highest risk of treatment failure, with high circulating IL-17A levels at diagnosis prognostic for persistent bacteremia and chronic tissue infection foci while a combination of IL-8 and CCL2 identifies increased mortality risk.
Objectives
To identify risk stratification biomarkers to enrich for the subset of Staphylococcus aureus bacteraemia patients who develop deep‐seated tissue infections with high morbidity and ...mortality to guide clinical trial enrolment and clinical management.
Methods
We evaluated the prognostic value of eight biomarkers for persistent bacteraemia, mortality and endovascular infection foci in a validation cohort of 160 patients with S. aureus bacteraemia enrolled consecutively over 3 years.
Results
High levels of IL‐17A, IL‐10 or soluble E‐selectin at bacteraemia diagnosis correlated with the duration of positive blood cultures. When thresholds defined in an independent cohort were applied, these biomarkers were robust predictors of persistent bacteraemia or endovascular infection. High serum levels of IL‐17A and IL‐10 often preceded the radiographic diagnosis of infective endocarditis, suggesting potential utility for prioritising diagnostic radiographic imaging. High IL‐8 was prognostic for all‐cause mortality, while IL‐17A and IL‐10 were superior to clinical metrics in discriminating between attributable mortality and non‐attributable mortality. High IL‐17A and IL‐10 identified more patients who developed microbiological failure or mortality than were identified by infective endocarditis diagnosis.
Conclusion
These biomarkers offer potential utility to identify patients at risk of persistent bacteraemia to guide diagnostic imaging and clinical management. Low biomarker levels could be used to rule out the need for more invasive TEE imaging in patients at lower risk of infective endocarditis. These biomarkers could enable clinical trials by enriching for patients with the greatest need for novel therapies.
We found that serum IL‐17A, IL‐10 and sE‐selectin are prognostic for persistent bacteraemia and infective endocarditis in patients hospitalised with S. aureus bacteraemia. High serum levels often preceded the radiographic diagnosis of infective endocarditis, suggesting potential utility for risk stratification. High IL‐17A identified more patients who meet clinical study endpoints of antibiotic failure than were identified by infective endocarditis diagnosis, offering potential utility as risk stratification biomarkers to facilitate patient selection for clinical trials.
Immunogenicity assessment during early stages of nonclinical biotherapeutic development is not always warranted. It is rarely predictive for clinical studies and evidence for the presence of ...anti-drug antibodies (ADAs) may be inferred from the pharmacokinetic (PK) profile. However, collecting and banking samples during the course of the study are prudent for confirmation and a deeper understanding of the impact on PK and safety. Biotherapeutic-specific ADA assays commonly developed can require considerable time and resources. In addition, the ADA assay may not be ready when needed if the study of PK and safety data triggers assay development. During early stages of drug development for antibody-drug conjugates (ADCs), there is the added complication of the potential inclusion of several molecular variants in a study, differing in the linker and/or drug components. To simplify analysis of ADAs at this stage, we developed plug-and-play generic approaches for both the assay format and the data analysis steps. Firstly, the assay format uses generic reagents to detect ADAs. Secondly, we propose a cut point methodology based on animal specific baseline variability instead of a population data approach. This assay showed good sensitivity, drug tolerance, and reproducibility across a variety of antibody-derived biotherapeutics without the need for optimization across molecules.
The 5th Workshop on Recent Issues in Bioanalysis (WRIB) was organized by the Calibration and Validation Group as a 2-day full immersion workshop for pharmaceutical companies, CROs and regulatory ...agencies to discuss, review, share perspectives, provide potential solutions and agree upon a consistent approach to recent issues in the bioanalysis of both small and large molecules. High quality, better compliance to regulations and scientific excellence are the foundation of this workshop. As in the previous editions of this significant event, recommendations were made and a consensus was reached among panelists and attendees, including industry leaders and regulatory experts representing the global bioanalytical community, on many 'hot' topics in bioanalysis. This 2011 White Paper is based on the conclusions from this workshop, and aims to provide a practical reference guide on those topics.
The measurement of therapeutic drug concentrations is used to assess drug exposure and the relationship between therapeutic pharmacokinetics (PK) and pharmacodynamics (PD), which help determine the ...optimal dose for patients. Ligand binding assays (LBAs) are often the method of choice for evaluation of drug concentration and use either the therapeutic target protein or antibodies to the therapeutic as capture and/or detection reagents. Due to the bivalency of antibody therapeutics, heterogeneous states of the drug/target complex can exist in the presence of soluble targets which can complicate measurement of unbound drug. In the case of bispecific antibodies, measurement of drug can be even more complicated and depend upon the levels of both targets to each arm. Measuring the total drug allows for PKPD modeling prediction of human dose projections in addition to overcoming challenges associated with measuring free drug for bispecific antibodies. Here, we present a study in which a sandwich ELISA format was used to measure total anti-KLK5/KLK7 antibody concentrations. This assay utilized a non-blocking anti-idiotype (ID) antibody to one arm of the antibody for capture and an antibody to target bound to the other arm of the antibody for detection. Our qualified assay showed acceptable precision, accuracy, dilutional linearity, and reproducibility and enabled detection of a total bispecific antibody at high levels of two targets. To confirm that our assay was detecting total drug, a subset of samples was evaluated in a generic total LC–MS/MS assay.
Graphical Abstract
causes serious bacterial infections with high morbidity and mortality, necessitating the discovery of new antibiotics. DSTA4637S is a novel antibody-antibiotic conjugate designed to target ...intracellular
that is not adequately eliminated by current standard-of-care antibiotics. DSTA4637S is composed of an anti-
Thiomab human immunoglobulin G1 (IgG1) monoclonal antibody linked to a novel rifamycin-class antibiotic (4-dimethylaminopiperidino-hydroxybenzoxazino rifamycin dmDNA31) via a protease-cleavable linker. Phagocytic cells ingest DSTA4637S-bound
, and intracellular cathepsins cleave the linker, releasing dmDNA31and killing intracellular
This first-in-human, randomized, double-blind, placebo-controlled, single-ascending-dose phase 1 trial analyzed the safety, pharmacokinetics, and immunogenicity of DSTA4637S in healthy volunteers. Thirty healthy male and female volunteers, 18-65 years old, were randomized into five cohorts receiving single intravenous (i.v.) doses of 5, 15, 50, 100, and 150 mg/kg of DSTA4637S or placebo (4 active:2 placebo). Subjects were followed for 85 days after dosing. No subject withdrew from the study, and no serious or severe adverse events occurred. One moderate infusion-related reaction (150 mg/kg DSTA4637S) occurred. No clinically meaningful or dose-related changes in laboratory parameters or vital signs occurred. Pharmacokinetics of plasma DSTA4637S conjugate and serum DSTA4637S total antibody were dose proportional. Systemic exposure of unconjugated dmDNA31 was low. No DSTA4637S-induced anti-drug antibody responses were observed. DSTA4637S was generally safe and well tolerated as a single i.v. dose in healthy volunteers. DSTA4637S has a favorable safety and pharmacokinetic profile that supports future development as a novel therapeutic for
infections. (This study has been registered at ClinicalTrials.gov under identifier NCT02596399.).
Macrophage colony stimulating factor 1 receptor (MCSF1R), osteopontin (OPN), high-mobility group protein B1 (HMGB1), glutamate dehydrogenase (GLDH), keratin 18 (K18), and caspase-cleaved keratin 18 ...(ccK18) are considered promising mechanistic biomarkers for the diagnosis of drug-induced liver injury. Here, we aim to elucidate the impact of the sample matrix and handling on the quantification of these emerging protein biomarkers. We investigated effects such as time from collection to centrifugation during serum (± gel) or EDTA plasma preparation on two assay platforms: immunoaffinity liquid chromatography mass spectrometric assays and sandwich immunoassays. Furthermore, we measured GLDH activity with an enzymatic activity assay. Matrix effects were observed particularly for HMGB1 and MCSF1R. HMGB1 levels were higher in serum than in plasma, whereas higher concentrations of MCSF1R were observed in plasma than in serum. A comparison of sample collection to centrifugation time ranging from 15 to 60 min demonstrated increasing levels of HMGB1 in serum, while MCSF1R, OPN, GLDH, and ccK18 concentrations remained stable. Additionally, there was a poor correlation in HMGB1 and ccK18 levels between serum and plasma. Considering the observed matrix effects, we recommend plasma as a matrix of choice and cross-study comparison studies to be limited to those using the same matrix.
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