Search

Summary: Background: Corticosteroids (CS) with or without adjuvant immunosuppressant agents are standard treatment for pemphigus vulgaris (PV). The efficacy of adjuvant therapies in minimizing steroid‐related adverse events (AEs) is unproven. Objectives: To utilize data collected in a French investigator‐initiated, phase III, open‐label, randomized controlled trial to demonstrate the efficacy and safety of rituximab and seek approval for its use in PV. Methods: This was an independently conducted post hoc analysis of the moderate‐to‐severe PV subset enrolled in the Ritux 3 study. Patients were randomized to rituximab plus 0·5 or 1·0 mg kg−1 per day prednisone tapered over 3 or 6 months, or 1·0 or 1·5 mg kg−1 per day prednisone alone tapered over 12 or 18 months, respectively (according to disease severity). The primary end point was complete remission at month 24 without CS (CRoff) for ≥ 2 months, and 24‐month efficacy and safety results were also reported. Results: At month 24, 34 of 38 patients (90%) on rituximab plus prednisone achieved CRoff ≥ 2 months vs. 10 of 36 patients (28%) on prednisone alone. Median total cumulative prednisone dose was 5800 mg in the rituximab plus prednisone arm vs. 20 520 mg for prednisone alone. Eight of 36 patients (22%) who received prednisone alone withdrew from treatment owing to AEs; one rituximab‐plus‐prednisone patient withdrew due to pregnancy. Overall, 24 of 36 patients (67%) on prednisone alone experienced a grade 3/4 CS‐related AE vs. 13 of 38 patients (34%) on rituximab plus prednisone. Conclusions: In patients with moderate‐to‐severe PV, rituximab plus short‐term prednisone was more effective than prednisone alone. Patients treated with rituximab had less CS exposure and were less likely to experience severe or life‐threatening CS‐related AEs. What's already known about this topic? Pemphigus vulgaris (PV) is the most common type of pemphigus.Corticosteroids, a standard first‐line treatment for PV, have significant side‐effects.Although their effects are unproven, adjuvant corticosteroid‐sparing agents are routinely used to minimize steroid exposure and corticosteroid‐related side‐effects.There is evidence that the anti‐CD20 antibody rituximab is effective in the treatment of patients with severe recalcitrant pemphigus and in patients with newly diagnosed pemphigus. What does this study add? This study provides a more detailed analysis of patients with PV enrolled in an investigator‐initiated trial.Rituximab plus prednisone had a steroid‐sparing effect and more patients achieved complete remission off prednisone.Fewer patients experienced grade 3 or grade 4 steroid‐related adverse events than those on prednisone alone.This collaboration between academia and industry, utilizing independent post hoc analyses, led to regulatory authority approvals of rituximab in moderate‐to‐severe PV. Linked Comment: Scorer et al. Br J Dermatol 2020; 182:1078–1079. [ABSTRACT FROM AUTHOR]

Target proteins that lack accessible binding pockets and conformational stability have posed increasing challenges for drug development. Induced proximity strategies, such as PROTACs and molecular glues, have thus gained attention as pharmacological alternatives, but still require small molecule docking at binding pockets for targeted protein degradation. The computational design of protein-based binders presents unique opportunities to access "undruggable" targets, but have often relied on stable 3D structures or structure-influenced latent spaces for effective binder generation. In this work, we introduce PepMLM , a target sequence-conditioned generator of de novo linear peptide binders. By employing a novel span masking strategy that uniquely positions cognate peptide sequences at the C-terminus of target protein sequences, PepMLM fine-tunes the state-of-the-art ESM-2 pLM to fully reconstruct the binder region, achieving low perplexities matching or improving upon validated peptide-protein sequence pairs. After successful in silico benchmarking with AlphaFold-Multimer, outperforming RFDiffusion on structured targets, we experimentally verify PepMLM's efficacy via fusion of model-derived peptides to E3 ubiquitin ligase domains, demonstrating endogenous degradation of emergent viral phosphoproteins and Huntington's disease-driving proteins. In total, PepMLM enables the generative design of candidate binders to any target protein, without the requirement of target structure, empowering downstream therapeutic applications., Competing Interests: Competing Interests P.C. and M.P.D. is a co-founder of UbiquiTx, Inc., and are inventors of patents related to genetically-encoded proteome editing technologies. The remaining authors declare no competing interests.

Immunogenicity assessment is an essential part of biotherapeutic drug development. While the immune response in animals is not always representative of the human immune response, immunogenicity data obtained in animal models is still informative for the evaluation of drug exposure and safety. The most common assay format used for the detection of anti-drug antibodies (ADAs) in preclinical and clinical studies is the bridging format. The advantage of this method is that it can detect all antibody isotypes generated against the therapeutic. However, the method development can be time-consuming and labor-intensive, due to the need for labeling of the drug which is used both as capture and detection. Various generic ADA assays have been successfully implemented to overcome these disadvantages and to enable faster assay development timelines to support nonclinical toxicology studies. Here, we describe the challenges in the development of an assay to detect antibodies to zinpentraxin alfa, a recombinant human pentraxin-2, in rabbit and rat toxicology studies. Our initial efforts to develop a bridging assay failed, prompting us to develop a method adapted from generic assay formats to detect anti-zinpentraxin alfa antibodies in the serum of different species with minimal optimization. However, while the general assay format remained similar, assay reagents were adapted between the different species, resulting in the development of two distinct assays for the detection of ADAs in rat and rabbit. Here, we share the final development/validation data and the immunogenicity study results. Our work highlights the need for the evaluation of alternate assay formats when evaluating novel drug modalities., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

MTBT 1466A is a monoclonal antibody designed to bind to mature human TGFβ3 in human tissue and systemic circulation. To evaluate binding of this therapeutic, a mature TGFβ3 assay was needed to be able to monitor pharmacodynamic responses in non-human primate (NHP) studies. However, mature TGFβ3 levels in systemic circulation are very low and require development of a highly sensitive assay for detection. This study describes the development of a highly sensitive, drug-tolerant pharmacodynamic biomarker assay for demonstrating target engagement in a pre-clinical study using MTBT1466A. Since mature TGFβ3 is a dimer, a single MAb was used as both the capture and detection antibodies. This assay was developed on the SMCxPRO platform and qualified based on current accepted criteria for biomarker assays. The assay demonstrated specificity to mature TGFβ3, with a lower limit of quantification of 31.3pg/mL. Although baseline levels of mature TGFβ3 were below the assay detection limit in 40% of animals within our study, 2- to 16-fold increases were observed in many of the animals following multiple-dosing regimen., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Anti-neutrophil cytoplasmic antibody-associated vasculitides granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) are rare, potentially organ- and life-threatening autoimmune conditions affecting adult and pediatric patients. An open-label phase II study was conducted to determine safe and effective dosing regimens of rituximab in pediatric patients with GPA/MPA. To determine the selection of an appropriate dose regimen in children for induction and maintenance, a population pharmacokinetic approach was used (nonlinear mixed-effect modeling), combining pediatric data with data from adults with GPA/MPA. The time course of B-cell depletion was assessed in both populations. The exposure-effect relationship was assessed by logistic regression. Twenty-five pediatric patients (80% female patients; age range, 6-17 years) were enrolled in the trial and received the induction regimen of intravenous rituximab 375 mg/m 2 weekly for 4 weeks, which resulted in a similar exposure to that of adults. Based on pharmacokinetic modeling, a maintenance dosing regimen of 250 mg/m 2 administered twice over 14 days followed by 250 mg/m 2 every 6 months is expected to result in similar rituximab exposure as that of adults receiving the approved maintenance dose of 500 mg administered twice over 14 days followed by 500 mg every 6 months. The time course of B-cell depletion was similar between the pediatric and adult populations, supporting the similarities in response in both populations and allowing extrapolation to patients less than 6 years old. Using a partial extrapolation approach helped identify safe and effective dosing regimens of rituximab in pediatric patients with GPA/MPA and lead to regulatory approval., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The T cell-dependent bispecific (TDB) antibody, anti-CD79b/CD3, targets CD79b and CD3 cell-surface receptors expressed on B cells and T cells, respectively. Since the anti-CD79b arm of this TDB binds only to human CD79b, a surrogate TDB that binds to cynomolgus monkey CD79b (cyCD79b) was used for preclinical characterization. To evaluate the impact of CD3 binding affinity on the TDB pharmacokinetics (PK), we utilized non-tumor-targeting bispecific anti-gD/CD3 antibodies composed of a low/high CD3 affinity arm along with a monospecific anti-gD arm as controls in monkeys and mice. An integrated PKPD model was developed to characterize PK and pharmacodynamics (PD). This study revealed the impact of CD3 binding affinity on anti-cyCD79b/CD3 PK. The surrogate anti-cyCD79b/CD3 TDB was highly effective in killing CD79b-expressing B cells and exhibited nonlinear PK in monkeys, consistent with target-mediated clearance. A dose-dependent decrease in B cell counts in peripheral blood was observed, as expected. Modeling indicated that anti-cyCD79b/CD3 TDB's rapid and target-mediated clearance may be attributed to faster internalization of CD79b, in addition to enhanced CD3 binding. The model yielded unbiased and precise curve fits. These findings highlight the complex interaction between TDBs and their targets and may be applicable to the development of other biotherapeutics.

The anti-immunoglobulin E (IgE) antibody, omalizumab (Xolair), is approved in the United States for the treatment of allergic asthma and chronic spontaneous urticaria, and has recently been studied for the treatment of nasal polyposis following completion of the two replicate phase 3 studies (POLYP 1 and POLYP 2). The dosing of omalizumab used in the phase 3 studies is based on a combination of patients' pre-treatment IgE level and body weight, similar to the approach used in allergic asthma. The objectives of the current analyses were to evaluate whether the pharmacokinetics (PK) of omalizumab and its pharmacodynamic (PD) effect on free and total IgE level in chronic rhinosinusitis with nasal polyps (CRSwNP) are consistent with those in allergic asthma via population PK/PD modeling and simulation, and to graphically explore exposure-response relationships and free IgE-response relationships in CRSwNP. Omalizumab PK and PD effect of total and free IgE in CRSwNP are generally consistent with those in asthma. Observed post-treatment free IgE suppressions were generally within the target range of the baseline IgE- and body weight-based omalizumab dosing table, with 74.2% and 93.0% of patients achieving a serum free IgE level below 25 ng/mL and 50 ng/mL, respectively at Week 24. Exposure-response analyses indicated that there was no clear correlation between omalizumab or free IgE concentration and key efficacy endpoints within the POLYP studies. Overall, these results indicate that the body weight and IgE-based dosing regimen of omalizumab was appropriate for use in CRSwNP patients., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CD20 is a cell-surface receptor expressed by healthy and neoplastic B cells and is a well-established target for biologics used to treat B-cell malignancies. Pharmacokinetic (PK) and pharmacodynamic (PD) data for the anti-CD20/CD3 T-cell-dependent bispecific antibody BTCT4465A were collected in transgenic mouse and nonhuman primate (NHP) studies. Pronounced nonlinearity in drug elimination was observed in the murine studies, and time-varying, nonlinear PK was observed in NHPs, where three empirical drug elimination terms were identified using a mixed-effects modeling approach: i) a constant nonsaturable linear clearance term (7 mL/day/kg); ii) a rapidly decaying time-varying, linear clearance term (t ½  = 1.6 h); and iii) a slowly decaying time-varying, nonlinear clearance term (t ½  = 4.8 days). The two time-varying drug elimination terms approximately track with time scales of B-cell depletion and T-cell migration/expansion within the central blood compartment. The mixed-effects NHP model was scaled to human and prospective clinical simulations were generated., (© 2018 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Objectives: To study the determinants of the pharmacokinetics (PK) of rituximab (RTX) in patients with ANCA-associated vasculitis (AAV) and its association with clinical outcomes., Methods: This study included data from 89 patients from the RTX in AAV trial who received the full dose of RTX (four weekly infusions of 375 mg/m2). RTX was quantified at weeks 2, 4, 8, 16 and 24, and summarized by computing the trapezoidal area under the curve. We explored potential determinants of the PK-RTX, and analysed its association with clinical outcomes: achievement of remission at 6 months, duration of B-cell depletion and time to relapse in patients who achieved complete remission., Results: RTX serum levels were significantly lower in males and in newly diagnosed patients, and negatively correlated with body surface area, baseline B-cell count and degree of disease activity. In multivariate analyses, the main determinants of PK-RTX were sex and new diagnosis. Patients reaching complete remission at month 6 had similar RTX levels compared with patients who did not reach complete remission. Patients with higher RTX levels generally experienced longer B-cell depletion than patients with lower levels, but RTX levels at the different time points and area under the curve were not associated with time to relapse., Conclusion: Despite the body-surface-area-based dosing protocol, PK-RTX is highly variable among patients with AAV, its main determinants being sex and newly diagnosed disease. We did not observe any relevant association between PK-RTX and clinical outcomes. The monitoring of serum RTX levels does not seem clinically useful in AAV.

Host cell proteins are manufacturing process-related impurities that may co-purify with the product despite extensive efforts to optimize the purification process. The risks associated with these impurities can vary and may be patient and/or therapeutic dependent. Therefore, it is critical to monitor and control the levels of these impurities in products and their potential impact on safety and efficacy. Lebrikizumab is a humanized immunoglobulin G4 monoclonal antibody (mAb) that binds specifically to soluble interleukin 13. This mAb is currently in phase III clinical development for the treatment of asthma. Following initial phase III studies, the material used in lebrikizumab clinical trials was found to have a process-related impurity identified as Chinese hamster ovary phospholipase B-like 2 (PLBL2) which co-purified with lebrikizumab. The immunogenic potential of PLBL2 and its potential impact on the immunogenicity of lebrikizumab in clinical studies were therefore evaluated. Data from the clinical studies demonstrated that ∼90% of subjects developed a specific and measurable immune response to PLBL2. Given the high incidence of antibodies to PLBL2 as well as the comparable safety profile observed between placebo- and drug-treated subjects, no correlation between safety events and anti-PLBL2 antibodies could be made. Additionally, no impact on the incidence of anti-lebrikizumab antibodies was observed, suggesting the lack of an adjuvant effect from PLBL2. Interim analysis from ongoing phase III studies using material with substantially reduced levels of PLBL2 with patients having had longer exposure shows significantly less and dose-dependent frequency of immune responses to PLBL2.

Aim: To improve on the efficiency of biomarker assay readiness, and for reliable biomarker data to support three drug programs, we implemented a fit-for-purpose approach, qualifying two biomarker assays and validating a third. Results/methodology: The qualification strategy and selection of experiments for two exploratory biomarkers (CXCL1, CCL19) was determined by the intended use of the biomarker data. The third biomarker, IL-6, was validated as the data would be used in monitoring patient safety during dose-escalation studies in a Phase I trial. All three assays passed a priori acceptance criteria., Conclusion: These assays highlight strategies and methodologies for a fit-for-purpose approach. Minimum qualification, full qualification and validation were chosen and supported programs at different stages of drug development.

Apo2L/TRAIL is a member of the tumor necrosis factor superfamily and an important inducer of apoptosis. Recombinant human (rhu) Apo2L/TRAIL has been attractive as a potential cancer therapeutic because many types of tumor cells are sensitive to its apoptosis-inducing effects. Nonclinical toxicology studies were conducted to evaluate the safety of rhuApo2L/TRAIL for possible use in humans. The cynomolgus monkey was chosen for this safety assessment based on high protein sequence homology between human and cynomolgus Apo2L/TRAIL and comparable expression of their receptors. Although hepatotoxicity was observed in repeat-dose monkey studies with rhuApo2L/TRAIL, all animals that displayed hepatotoxicity had developed antitherapeutic antibodies (ATAs). The cynomolgus ATAs augmented the cytotoxicity of rhuApo2L/TRAIL but not of its cynomolgus counterpart. Of note, human and cynomolgus Apo2L/TRAIL differ by four amino acids, three of which are surface-exposed. In vivo studies comparing human and cynomolgus Apo2L/TRAIL supported the conclusion that these distinct amino acids served as epitopes for cross-species ATAs, capable of crosslinking rhuApo2L/TRAIL and thus triggering hepatocyte apoptosis. We describe a hapten-independent mechanism of immune-mediated, drug-related hepatotoxicity - in this case - associated with the administration of a human recombinant protein in monkeys. The elucidation of this mechanism enabled successful transition of rhuApo2L/TRAIL into human clinical trials.

Therapeutic monoclonal immunoglobulins (mAbs) are used to treat patients with a wide range of disorders including autoimmune diseases. As pharmaceutical companies bring more fully humanized therapeutic mAb drugs to the healthcare market analytical platforms that perform therapeutic drug monitoring (TDM) without relying on mAb specific reagents will be needed. In this study we demonstrate that liquid-chromatography-mass spectrometry (LC-MS) can be used to perform TDM of mAbs in the same manner as smaller nonbiologic drugs. The assay uses commercially available reagents combined with heavy and light chain disulfide bond reduction followed by light chain analysis by microflow-LC-electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS). Quantification is performed using the peak areas from multiply charged mAb light chain ions using an in-house developed software package developed for TDM of mAbs. The data presented here demonstrate the ability of an LC-MS assay to quantify a therapeutic mAb in a large cohort of patients in a clinical trial. The ability to quantify any mAb in serum via the reduced light chain without the need for reagents specific for each mAb demonstrates the unique capabilities of LC-MS. This fact, coupled with the ability to phenotype a patient's polyclonal repertoire in the same analysis further shows the potential of this approach to mAb analysis.

Introduction: In a subset of patients with asthma, standard-of-care treatment does not achieve disease control, highlighting the need for novel therapeutic approaches. Lebrikizumab is a humanised, monoclonal antibody that binds to and blocks interleukin-13 activity., Methods: LUTE and VERSE were replicate, randomised, double-blind, placebo-controlled studies, evaluating multiple doses of lebrikizumab in patients with uncontrolled asthma despite the use of medium-to-high-dose inhaled corticosteroid and a second controller. Patients received lebrikizumab 37.5, 125, 250 mg or placebo subcutaneously every four weeks. The primary endpoint was the rate of asthma exacerbations during the placebo-controlled period. Analyses were performed on prespecified subgroups based on baseline serum periostin levels. Following the discovery of a host-cell impurity in the study drug material, protocols were amended to convert from phase III to phase IIb. Subsequently, dosing of study medication was discontinued early as a precautionary measure. The data collected for analysis were from a placebo-controlled period of variable duration and pooled across both studies., Results: The median duration of treatment was approximately 24 weeks. Treatment with lebrikizumab reduced the rate of asthma exacerbations, which was more pronounced in the periostin-high patients (all doses: 60% reduction) than in the periostin-low patients (all doses: 5% reduction); no dose-response was evident. Lung function also improved following lebrikizumab treatment, with greatest increase in FEV1 in periostin-high patients (all doses: 9.1% placebo-adjusted improvement) compared with periostin-low patients (all doses: 2.6% placebo-adjusted improvement). Lebrikizumab was well tolerated and no clinically important safety signals were observed., Conclusions: These data are consistent with, and extend, previously published results demonstrating the efficacy of lebrikizumab in improving rate of asthma exacerbations and lung function in patients with moderate-to-severe asthma who remain uncontrolled despite current standard-of-care treatment., Trial Registration Numbers: The LUTE study was registered under NCT01545440 and the VERSE study under NCT01545453 at http://www.clinicaltrials.gov., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Background: The anti-IgE monoclonal antibody, omalizumab, is approved in the US as add-on therapy for patients ≥12 years of age with moderate-to-severe persistent allergic asthma. Omalizumab is administered according to the US Food and Drug Administration approved dosing table included in the prescribing information. The dosing table was developed using Genentech's free IgE assay and is designed to achieve free serum IgE levels of <50 ng/mL, known to be associated with clinical benefit. Lack of clinical benefit in a subset of patients on omalizumab has prompted demand for commercial free IgE assays to guide omalizumab dosing. To date, two commercial free IgE assays marketed by ViraCor-IBT (no longer offered) and BioTeZ have been available to physicians., Objective: This study compares the results generated from the two commercial free IgE assays with the free IgE levels generated by the Genentech assay., Methods: Two serum sample sets were prepared using 20 samples from patients with a wide range of IgE and omalizumab from an omalizumab clinical trial and 36 samples from omalizumab-naïve patients. Different amounts of omalizumab were added to the 36 omalizumab naïve samples based on measured total IgE levels to ensure that a good range of IgE and omalizumab was represented in the study samples. Samples were randomized for blinded analysis of free IgE levels using the Genentech, ViraCor-IBT and BioTeZ free serum IgE assays. Analysis of samples in the ViraCor-IBT assay were conducted by ViraCor-IBT and the analysis of samples using the Genentech and BioTeZ assay methods were conducted by a third party contract research organization., Results: The ViraCor-IBT and BioTeZ free IgE assays demonstrated significantly higher free IgE levels than the Genentech free IgE assay. Twenty-nine of 56 samples tested <50 ng/mL in the Genentech assay; of these, 12/29 (41%) and 20/29 (69%) tested >50 ng/mL in the BioTeZ and ViraCor-IBT assays, respectively. In the BioTeZ free IgE evaluations, 11/20 samples that were re-tested had inter-assay differences ranging from 40-190%., Conclusions: Free ligand (such as IgE) measurements are challenging and dependent on the method and reagents used. The Viracor-IBT and BioTeZ methods tend to over-estimate free serum IgE levels compared with the Genentech free IgE assay. Using these assays to monitor therapy and adjust omalizumab doses post treatment is considered off-label use and could lead to a potential risk for unnecessary treatment and/or risk to patient safety.

HAE1, a high-affinity anti-IgE monoclonal antibody, is discussed here as a case study in the use of quantitative pharmacology in the development of a second-generation molecule. In vitro, preclinical, and clinical data from the first-generation molecule, omalizumab, were heavily leveraged in the HAE1 program. A preliminary mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for HAE1 was developed using an existing model for omalizumab, together with in vitro binding data for HAE1 and omalizumab. When phase I data were available, the model was refined by simultaneously modeling PK/PD data from omalizumab studies with the available HAE1 phase I data. The HAE1 clinical program was based on knowledge of the quantitative relationship between a pharmacodynamic biomarker, suppression of free IgE, and clinical response (e.g., lower exacerbation rates) obtained in pivotal studies with omalizumab. A clinical trial simulation platform was developed to predict free IgE levels and clinical responses following attainment of a target free IgE level (