Evaluation of an AAV9-mini-dystrophin gene therapy candidate in a rat model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the dystrophin gene and is characterized by muscle wasting and early mortality. Adeno-associated virus-mediated gene therapy is being investigated as a treatment for DMD. In the nonclinical study documented here, we determined the effective dose of fordadistrogene movaparvovec, a clinical candidate adeno-associated virus serotype 9 vector carrying a human mini-dystrophin transgene, after single intravenous injection in a dystrophin-deficient (DMDmdx) rat model of DMD. Overall, we found that transduction efficiency, number of muscle fibers expressing the human mini-dystrophin polypeptide, improvement of the skeletal and cardiac muscle tissue architecture, correction of muscle strength and fatigability, and improvement of diastolic and systolic cardiac function were directly correlated with the amount of vector administered. The effective dose was then tested in older DMDmdx rats with a more dystrophic phenotype similar to the pathology observed in older patients with DMD. Except for a less complete rescue of muscle function in the oldest cohort, fordadistrogene movaparvovec was also found to be therapeutically effective in older DMDmdx rats, suggesting that this product may be appropriate for evaluation in patients with DMD at all stages of disease.

Dystrophin and mini-dystrophin quantification by mass spectrometry in skeletal muscle for gene therapy development in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disorder caused by mutations in the DMD gene, leading to severe reduction or absence of the protein dystrophin. Gene therapy strategies that aim to increase expression of a functional dystrophin protein (mini-dystrophin) are under investigation. The ability to accurately quantify dystrophin/mini-dystrophin is essential in assessing the level of gene transduction. We demonstrated the validation and application of a novel peptide immunoaffinity liquid chromatography-tandem mass spectrometry (IA-LC-MS/MS) assay. Data showed that dystrophin expression in Becker muscular dystrophy and DMD tissues, normalized against the mean of non-dystrophic control tissues (n = 20), was 4-84.5% (mean 32%, n = 20) and 0.4-24.1% (mean 5%, n = 20), respectively. In a DMD rat model, biceps femoris tissue from dystrophin-deficient rats treated with AAV9.hCK.Hopti-Dys3978.spA, an adeno-associated virus vector containing a mini-dystrophin transgene, showed a dose-dependent increase in mini-dystrophin expression at 6 months post-dose, exceeding wildtype dystrophin levels at high doses. Validation data showed that inter- and intra-assay precision were ≤20% (≤25% at the lower limit of quantification [LLOQ]) and inter- and intra-run relative error was within ±20% (±25% at LLOQ). IA-LC-MS/MS accurately quantifies dystrophin/mini-dystrophin in human and preclinical species with sufficient sensitivity for immediate application in preclinical/clinical trials.

A Mechanistic Site-Of-Action Model: A Tool for Informing Right Target, Right Compound, And Right Dose for Therapeutic Antagonistic Antibody Programs.

Quantitative modeling is increasingly utilized in the drug discovery and development process, from the initial stages of target selection, through clinical studies. The modeling can provide guidance on three major questions-is this the right target, what are the right compound properties, and what is the right dose for moving the best possible candidate forward. In this manuscript, we present a site-of-action modeling framework which we apply to monoclonal antibodies against soluble targets. We give a comprehensive overview of how we construct the model and how we parametrize it and include several examples of how to apply this framework for answering the questions postulated above. The utilities and limitations of this approach are discussed.

Human FcRn Tissue Expression Profile and Half-Life in PBMCs.

System-wide quantitative characterization of human neonatal Fc receptor (FcRn) properties is critical for understanding and predicting human PK (pharmacokinetics) as well as the distribution of mAbs and Fc-fusion proteins using PBPK (physiologically-based pharmacokinetic) modeling. To this end, tissue-specific FcRn expression and half-life are important model inputs. Herein, human FcRn tissue expression was measured by peptide immunoaffinity chromatography coupled with high-resolution mass spectrometry. FcRn concentrations across 14 human tissues ranged from low to 230 pmol per gram of tissue. Furthermore, the FcRn half-life was determined to be 11.1 h from a human stable isotope labelled leucine pulse labeling experiment. The spatial and temporal quantitative human FcRn data now promise to enable a refined PBPK model with improved accuracy of human PK predictions for Fc-containing biotherapeutics.

Assessing the Feasibility of Neutralizing Osteopontin with Various Therapeutic Antibody Modalities.

Osteopontin is a secreted glycophosphoprotein that is highly implicated in many physiological and pathological processes such as biomineralization, cell-mediated immunity, inflammation, fibrosis, cell survival, tumorigenesis and metastasis. Antibodies against osteopontin have been actively pursued as potential therapeutics for various diseases by pharmaceutical companies and academic laboratories. Many studies have demonstrated the efficacy of osteopontin inhibition in a variety of preclinical models of diseases such as rheumatoid arthritis, cancer, nonalcoholic steatohepatitis, but clinical utility has not yet been demonstrated. To evaluate the feasibility of osteopontin neutralization with antibodies in a clinical setting, we measured its physiological turnover rate in humans, a sensitive parameter required for mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Results from a stable isotope-labelled amino acid pulse-chase study in healthy human subjects followed by mass spectrometry showed that osteopontin undergoes very rapid turnover. PK/PD modeling and simulation of different theoretical scenarios reveal that achieving sufficient target coverage using antibodies can be very challenging mostly due to osteopontin's fast turnover, as well as its relatively high plasma concentrations in human. Therapeutic antibodies against osteopontin would need to be engineered to have much extended PK than conventional antibodies, and be administered at high doses and with short dosing intervals.

Biomeasures and mechanistic modeling highlight PK/PD risks for a monoclonal antibody targeting Fn14 in kidney disease.

Discovery of the upregulation of fibroblast growth factor-inducible-14 (Fn14) receptor following tissue injury has prompted investigation into biotherapeutic targeting of the Fn14 receptor for the treatment of conditions such as chronic kidney diseases. In the development of monoclonal antibody (mAb) therapeutics, there is an increasing trend to use biomeasures combined with mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modeling to enable decision making in early discovery. With the aim of guiding preclinical efforts on designing an antibody with optimized properties, we developed a mechanistic site-of-action (SoA) PK/PD model for human application. This model incorporates experimental biomeasures, including concentration of soluble Fn14 (sFn14) in human plasma and membrane Fn14 (mFn14) in human kidney tissue, and turnover rate of human sFn14. Pulse-chase studies using stable isotope-labeled amino acids and mass spectrometry indicated the sFn14 half-life to be approximately 5 hours in healthy volunteers. The biomeasures (concentration, turnover) of sFn14 in plasma reveals a significant hurdle in designing an antibody against Fn14 with desired characteristics. The projected dose (>1 mg/kg/wk for 90% target coverage) derived from the human PK/PD model revealed potential high and frequent dosing requirements under certain conditions. The PK/PD model suggested a unique bell-shaped relationship between target coverage and antibody affinity for anti-Fn14 mAb, which could be applied to direct the antibody engineering towards an optimized affinity. This investigation highlighted potential applications, including assessment of PK/PD risks during early target validation, human dose prediction and drug candidate optimization.

Protein Turnover Measurements in Human Serum by Serial Immunoaffinity LC-MS/MS.

BACKGROUND: The half-life of target proteins is frequently an important parameter in mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Clinical studies for accurate measurement of physiologically relevant protein turnover can reduce the uncertainty in PK/PD model-based predictions, for example, of the therapeutic dose and dosing regimen in first-in-human clinical trials. METHODS: We used a targeted mass spectrometry work flow based on serial immunoaffinity enrichment ofmultiple human serum proteins from a [5,5,5-2H3]-L-leucine tracer pulse-chase study in healthy volunteers. To confirm the reproducibility of turnover measurements from serial immunoaffinity enrichment, multiple aliquots from the same sample set were subjected to protein turnover analysis in varying order. Tracer incorporation was measured by multiple-reaction-monitoring mass spectrometry and target turnover was calculated using a four-compartment pharmacokinetic model. RESULTS: Five proteins of clinical or therapeutic relevance including soluble tumor necrosis factor receptor superfamily member 12A, tissue factor pathway inhibitor, soluble interleukin 1 receptor like 1, soluble mucosal addressin cell adhesion molecule 1, and muscle-specific creatine kinase were sequentially subjected to turnover analysis from the same human serum sample. Calculated half-lives ranged from 5-15 h; however, no tracer incorporation was observed for mucosal addressin cell adhesion molecule 1. CONCLUSIONS: The utility of clinical pulse-chase studies to investigate protein turnover can be extended by serial immunoaffinity enrichment of target proteins. Turnover analysis from serum and subsequently from remaining supernatants provided analytical sensitivity and reproducibility for multiple human target proteins in the same sample set, irrespective of the order of analysis.

Scaling Proteome-Wide Reactions of Activity-Based Probes.

Unified analysis of complex reactions of an activity-based probe with proteins in a proteome remains an unsolved challenge. We propose a power expression, rate = kobs[Probe]α, for scaling the progress of proteome-wide reactions and use the scaling factor (0 ≤ α ≤ 1) as an apparent, partial order with respect to the probe to measure the "enzyme-likeness" for a protein in reaction acceleration. Thus, α reports the intrinsic reactivity of the protein with the probe. When α = 0, the involved protein expedites the reaction to the maximal degree; when α = 1, the protein reacts with the probe via an unaccelerated, bimolecular reaction. The selectivity (ß) of the probe reacting with two proteins is calculated as a ratio of conversion factors (kobs values) for corresponding power equations. A combination of α and ß provides a tiered system for quantitatively assessing the probe efficacy; an ideal probe exhibits high reactivity with its protein targets (low in α) and is highly selective (high in ß) in forming the probe-protein adducts. The scaling analysis was demonstrated using proteome-wide reactions of HT-29 cell lysates with a model probe of threonine ß-lactone.

Development of structural marker peptides for cystic fibrosis transmembrane conductance regulator in cell plasma membrane by reversed-footprinting mass spectrometry.

A targeted mass spectrometry-based method is presented that adopts the fast photochemical oxidation of proteins (FPOP) for footprinting of cystic fibrosis transmembrane conductance regulator (CFTR) membrane transporter at its original plasma membrane location. Two analytical imperatives were sought: (1) overall simplification in data acquisition and analysis and (2) lower quantitation limits, which enabled direct analysis of intrinsically low-abundance transmembrane proteins. These goals were achieved by using a reversed-footprinting technique that monitored the unoxidized peptides remaining after the FPOP treatment. In searching for structurally informative peptides, a workflow was designed for accurate and precise quantitation of CFTR peptides produced from proteolytically digesting the plasma membrane subproteome of cells. This sample preparation strategy mitigated the need for challenging purification of large quantities of structurally intact CFTR. On the basis of the interrogated peptides, it was proposed a concept of the structural marker peptide that could report CFTR structure and function in cells. The reversed-footprinting mass spectrometry extends the FPOP technology to study conformation and interaction changes of low-abundance proteins directly in their endogenous cellular locations.

Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane.

Deficient chloride transport through cystic fibrosis (CF) transmembrane conductance regulator (CFTR) causes lethal complications in CF patients. CF is the most common autosomal recessive genetic disease, which is caused by mutations in the CFTR gene; thus, CFTR mutants can serve as primary targets for drugs to modulate and rescue the ion channel's function. The first step of drug modulation is to increase the expression of CFTR in the apical plasma membrane (PM); thus, accurate measurement of CFTR in the PM is desired. This work reports a tandem enrichment strategy to prepare PM CFTR and uses a stable isotope labeled CFTR sample as the quantitation reference to measure the absolute amount of apical PM expression of CFTR in CFBE 41o- cells. It was found that CFBE 41o- cells expressing wild-type CFTR (wtCFTR), when cultured on plates, had 2.9 ng of the protein in the apical PM per million cells; this represented 10% of the total CFTR found in the cells. When these cells were polarized on filters, the apical PM expression of CFTR increased to 14%. Turnover of CFTR in the apical PM of baby hamster kidney cells overexpressing wtCFTR (BHK-wtCFTR) was also quantified by targeted proteomics based on multiple reaction monitoring mass spectrometry; wtCFTR had a half-life of 29.0 ± 2.5 h in the apical PM. This represents the first direct measurement of CFTR turnover using stable isotopes. The absolute quantitation and turnover measurements of CFTR in the apical PM can significantly facilitate understanding the disease mechanism of CF and thus the development of new disease-modifying drugs. Absolute CFTR quantitation allows for direct result comparisons among analyses, analysts, and laboratories and will greatly amplify the overall outcome of CF research and therapy.

Stable isotope dilution mass spectrometry for membrane transporter quantitation.

This review provides an introduction to stable isotope dilution mass spectrometry (MS) and its emerging applications in the analysis of membrane transporter proteins. Various approaches and application examples, for the generation and use of quantitation reference standards­either stable isotope-labeled peptides or proteins­are discussed as they apply to the MS quantitation of membrane proteins. Technological considerations for the sample preparation of membrane transporter proteins are also presented.

Serine lipids of Porphyromonas gingivalis are human and mouse Toll-like receptor 2 ligands.

The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2(-/-)) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2(-/-) mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

Bacterial lipodipeptide, Lipid 654, is a microbiome-associated biomarker for multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disease of unknown etiology. Infectious agents have been suggested to have a role as environmental factors in MS, but this concept remains controversial. Recently, gastrointestinal commensal bacteria have been implicated in the pathogenesis of autoimmune diseases, but mechanisms underlying the relationship of human systemic autoimmunity with the commensal microbiome have yet to be identified. Consistent with the lack of understanding of pathogenic mechanisms and relevant environmental factors in MS, no blood biomarkers have been identified that distinguish MS patients from healthy individuals. We recently identified a unique gastrointestinal and oral bacteria-derived lipodipeptide, Lipid 654, which is produced by commensal bacteria and functions as a human and mouse Toll-like receptor 2 ligand. Using multiple-reaction-monitoring mass spectrometry, a critical approach in targeted lipidomics, we now report that Lipid 654 can be recovered in the serum of healthy individuals. Most interestingly, we find that Lipid 654 is expressed at significantly lower levels in the serum of patients with MS compared with both healthy individuals and patients with Alzheimer's disease. These results thus identify for the first time a potential mechanism relating the gastrointestinal and oral commensal microbiome to a human systemic autoimmune disease. In addition, these results also identify a potential etiologic environmental factor and novel clinically relevant serum biomarker for MS.