Pharmacokinetics and pharmacodynamics of Abelacimab (MAA868), a novel dual inhibitor of Factor XI and Factor XIa.

BACKGROUND: Factor XI (FXI) inhibition offers the promise of hemostasis-sparing anticoagulation for the prevention and treatment of thromboembolic events. Abelacimab (MAA868) is a novel fully human monoclonal antibody that targets the catalytic domain and has dual activity against the inactive zymogen Factor XI and the activated FXI. OBJECTIVES: To investigate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of single dose intravenous and multiple dose subcutaneous administration of abelacimab in healthy volunteers and patients with atrial fibrillation, respectively. PATIENTS/METHODS: In study ANT-003, healthy volunteers were administered single intravenous doses of abelacimab (30-150 mg) or placebo. The ANT-003 study also included a cohort of obese but otherwise healthy subjects. In study ANT-004, patients with atrial fibrillation were administered monthly subcutaneous doses of abelacimab (120 mg and 180 mg), or placebo, for 3 months. Key PK and PD parameters, including activated partial thromboplastin time (aPTT) and free FXI levels, as well as anti-drug antibodies (ADA) were assessed. RESULTS: Following intravenous administration of abelacimab, the terminal elimination half-life ranged from 25 to 30 days. One hour after the start of the intravenous infusion greater than 99% reductions in free FXI levels were observed. Following once monthly subcutaneous administration, marked reductions from baseline in free FXI levels were sustained. Parenteral administration of abelacimab demonstrated a favorable safety profile with no clinically relevant bleeding events. CONCLUSIONS: Intravenous and multiple subcutaneous dose administration of abelacimab were safe and well tolerated. The safety, PK, and PD data from these studies support the clinical development of abelacimab.

Biophysical and Immunological Characterization and In Vivo Pharmacokinetics and Toxicology in Nonhuman Primates of the Anti-PD-1 Antibody Pembrolizumab.

The programmed cell death 1 (PD-1) pathway represents a major immune checkpoint, which may be engaged by cells in the tumor microenvironment to overcome active T-cell immune surveillance. Pembrolizumab (Keytruda®, MK-3475) is a potent and highly selective humanized mAb of the IgG4/kappa isotype designed to directly block the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of T cells to facilitate tumor regression and ultimately immune rejection. Pembrolizumab binds to human and cynomolgus monkey PD-1 with picomolar affinity and blocks the binding of human and cynomolgus monkey PD-1 to PD-L1 and PD-L2 with comparable potency. Pembrolizumab binds both the C'D and FG loops of PD-1. Pembrolizumab overcomes human and cynomolgus monkey PD-L1-mediated immune suppression in T-cell cultures by enhancing IL2 production following staphylococcal enterotoxin B stimulation of healthy donor and cancer patient cells, and IFNγ production in human primary tumor histoculture. Ex vivo and in vitro studies with human and primate T cells show that pembrolizumab enhances antigen-specific T-cell IFNγ and IL2 production. Pembrolizumab does not mediate FcR or complement-driven effector function against PD-1-expressing cells. Pembrolizumab displays dose-dependent clearance and half-life in cynomolgus monkey pharmacokinetic and toxicokinetic studies typical for human IgG4 antibodies. In nonhuman primate toxicology studies, no findings of toxicologic significance were observed. The preclinical data for pembrolizumab are consistent with the clinical anticancer activity and safety that has been demonstrated in human clinical trials.

A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells.

Yeast cells, in particular Pichia pastoris, are the host cell of choice for manufacturing several protein therapeutic agents in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and the residual DNA after the purification process of the drug must be monitored to ensure drug purity and safety. Currently, real-time PCR (qPCR) based methods are widely employed for quantification of host residual DNA. At the same time the digital PCR technology is coming into prominence with promise of higher sensitivity. Here we report a method where the protein drug is directly added to the droplet digital PCR (ddPCR) reaction including yeast-specific primers and fluorescent-tagged probe and nanoliter-sized droplets are generated. The droplets are then subjected to PCR followed by analysis for fluorescence. This Pichia residual DNA direct ddPCR method for yeast can be used to test higher amount of drug compared to the corresponding qPCR method thereby increasing sensitivity, retaining high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with three batches of a recombinant human IgG1-Fc-based drug (RP-1) and with commercially available human insulin, both manufactured in yeast cells. This method simplifies the residual DNA quantification protocol by eliminating DNA extraction or protease digestion and eliminates use of DNA standards in day-to-day running of the method.

Phase I trial of intravesical recombinant adenovirus mediated interferon-α2b formulated in Syn3 for Bacillus Calmette-Guérin failures in nonmuscle invasive bladder cancer.

PURPOSE: A phase I trial of intravesical recombinant adenovirus mediated interferon-α2b gene therapy (rAd-IFNα) formulated with the excipient SCH Syn3 was conducted in patients with nonmuscle invasive bladder cancer who had disease recurrence after treatment with bacillus Calmette-Guérin. The primary objective was to determine the safety of rAd-IFNα/Syn3. Secondary end points were demonstrated effective rAd-IFNα gene expression and preliminary evidence of clinical activity at 3 months. MATERIALS AND METHODS: A total of 17 patients with recurrent nonmuscle invasive bladder cancer after bacillus Calmette-Guérin treatment were enrolled in the study. A single treatment of rAd-IFNα (3 × 10(9) to 3 × 10(11) particles per ml) formulated with the excipient Syn3 was administered. Patient safety was evaluated for 12 or more weeks. Efficacy of gene transfer was determined by urine IFNα protein concentrations. Preliminary drug efficacy was determined at 3 months. RESULTS: Intravesical rAd-IFNα/Syn3 was well tolerated as no dose limiting toxicity was encountered. Urgency was the most common adverse event and all cases were grade 1 or 2. rAd-IFNα DNA was not detected in the blood. However, transient low serum IFNα and Syn3 levels were measured. High and prolonged dose related urine IFNα levels were achieved with the initial treatment. Of the 14 patients treated at doses of 10(10) or more particles per ml with detectable urine IFNα, 6 (43%) experienced a complete response at 3 months and 2 remained disease-free at 29.0 and 39.2 months, respectively. CONCLUSIONS: Intravesical rAd-IFNα/Syn3 was well tolerated with no dose limiting toxicity encountered. Dose dependent urinary IFNα concentrations confirmed efficient gene transfer and expression. Intravesical rAd-IFNα/Syn3 demonstrated clinical activity in nonmuscle invasive bladder cancer recurring after bacillus Calmette-Guérin.

Challenges of developing and validating immunogenicity assays to support comparability studies for biosimilar drug development.

Imminent patent expiry for a number of biological products currently on the market (many of which are blockbusters) has created an increasing opportunity for the development of biosimilars in the biotechnology industry. The key for successful biosimilar development is to demonstrate biosimilarity to the originator drug. In addition to demonstrating the similarity of physical and chemical properties between biosimilar and originator compounds, regulatory agencies require that immunogenicity be evaluated in comparative studies between biosimilar and originator drugs. Immunogenicity assays are generally non-quantitative (qualitative) and proving similarity/comparability based on qualitative assays can be very challenging. This review will discuss the challenges of developing and validating immunogenicity assays to support preclinical and clinical comparative studies for biosimilar drug development as well as the challenges in association with the interpretation of the data.

Recommendations and requirements for the design of bioanalytical testing used in comparability studies for biosimilar drug development.

With the imminent expiry of patents on a number of biological products on the market, the development of biosimilars (or 'follow-on biologics') creates an increasing opportunity in the biotechnology industry. Although general guidelines on the quality and safety of biological products also apply to biosimilars, there is a need to address specific requirements for developing biosimilar drugs. Since it is critical to show comparability of the biosimilar products to their reference (or innovator) products, developing the appropriate bioanalytical methods to support such preclinical and clinical comparability studies is of great importance. The present work recommends the requirements for the development and validation for both pharmacokinetic and immunogenicity assays to support the biosimilar drug development.

Identification of HCV protease inhibitor resistance mutations by selection pressure-based method.

A major challenge to successful antiviral therapy is the emergence of drug-resistant viruses. Recent studies have developed several automated analyses of HIV sequence polymorphism based on calculations of selection pressure (K(a)/K(s)) to predict drug resistance mutations. Similar resistance analysis programs for HCV inhibitors are not currently available. Taking advantage of the recently available sequence data of patient HCV samples from a Phase II clinical study of protease inhibitor boceprevir, we calculated the selection pressure for all codons in the HCV protease region (amino acid 1-181) to identify potential resistance mutations. The correlation between mutations was also calculated to evaluate linkage between any two mutations. Using this approach, we identified previously known major resistant mutations, including a recently reported mutation V55A. In addition, a novel mutation V158I was identified, and we further confirmed its resistance to boceprevir in protease enzyme and replicon assay. We also extended the approach to analyze potential interactions between individual mutations and identified three pairs of correlated changes. Our data suggests that selection pressure-based analysis and correlation mapping could provide useful tools to analyze large amount of sequencing data from clinical samples and to identify new drug resistance mutations as well as their linkage and correlations.

SCH 412499: biodistribution and safety of an adenovirus containing P21(WAF-1/CIP-1) following subconjunctival injection in Cynomolgus monkeys.

Monkey studies were conducted for the preclinical safety assessment of SCH 412499, an adenovirus encoding p21, administered by subconjunctival injection prior to trabeculectomy for postoperative maintenance of the surgical opening. Biodistribution of SCH 412499 was minimal and there was no systemic toxicity. Findings included swollen, partially closed or shut eye(s) and transient congestion in the conjunctiva. A mononuclear cell infiltrate was present in the conjunctiva, choroid and other ocular tissues, but completely or partially resolved over time. Electroretinograms and visual evoked potentials revealed no adverse findings. Thus, the findings are not expected to preclude the clinical investigation of SCH 412499.

Pharmacodynamics of PEG-IFN alpha differentiate HIV/HCV coinfected sustained virological responders from nonresponders.

Pegylated interferon (PEG-IFN) has become standard therapy for hepatitis C virus (HCV) infection. We evaluated whether PEG-IFN pharmacodynamics and pharmacokinetics account for differences in treatment outcome and whether these parameters might be predictors of therapeutic outcome. Twenty-four IFN-naïve, HCV/human immunodeficiency virus-coinfected patients received PEG-IFN alpha-2b (1.5 microg/kg) once weekly plus daily ribavirin (1000 or 1200 mg) for up to 48 weeks. HCV RNA and PEG-IFN alpha concentrations were obtained from samples collected frequently after the first 3 PEG-IFN doses. We modeled HCV kinetics incorporating pharmacokinetic and pharmacodynamic parameters. Although PEG-IFN concentrations and pharmacokinetic parameters were similar in sustained virological responders (SVRs) and nonresponders (NRs), the PEG-IFN alpha-2b concentration that decreases HCV production by 50% (EC50) was lower in SVRs compared with NRs (0.04 vs. 0.45 microg/L [P = .014]). Additionally, the median therapeutic quotient (i.e., the ratio between average PEG-IFN concentration and EC50 [C/EC50]), and the PEG-IFN concentration at day 7 divided by EC50 (C(7)/EC50) were significantly increased in SVRs compared with NRs after the first (10.1 vs. 1.0 [P = .012], 2.8 vs. 0.3 [P = .007], respectively) and second (14.0 vs. 1.1 [P = .016], 5.4 vs. 0.4 [P = .02], respectively) PEG-IFN doses. All 3 parameters may be used to identify NRs. In conclusion, PEG-IFN concentrations and pharmacokinetic parameters do not differ between SVRs and NRs. In contrast, pharmacodynamic measurements-namely EC50, the therapeutic quotient, and C(7)/EC50--are different in coinfected SVRs and NRs. These parameters might be useful predictors of treatment outcome during the first month of therapy.

Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway.

Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.

Development and validation of a kinetic assay for analysis of anti-human interleukin-5 monoclonal antibody (SCH 55700) and human interleukin-5 interactions using surface plasmon resonance.

A method to assess the kinetic interactions of a humanized anti-human interleukin-5 (IL-5) monoclonal antibody (SCH 55700) with native human IL-5 using surface plasmon resonance (SPR) has been developed and validated. Since there are no clearly defined validation requirements for a SPR-based binding kinetic assay, the validation strategy was based on the guidelines stipulated by the International Conference on Harmonization for Analytical Method Validation. Due to the uniqueness of the method, however, proper interpretation of the guidance was critical for establishing a validation plan. Validation was designed to assess repeatability, intermediate precision, specificity, linearity, and robustness which included analysis of baseline stability and reproducibility of ligand immobilization. Additionally, system suitability criteria were established to assure that the assay consistently performs as it was intended. The experimental artifacts that can complicate kinetic analysis using biosensor technology, such as heterogeneity of the ligand, mass transport, and nonspecific binding, were considered during the development of this assay. For each run, replicate concentrations of SCH 55700 were injected randomly over the immobilized surfaces to acquire association- and dissociation-phase data. The data were transformed and double referenced to remove systematic deviations seen in the binding responses. Association and dissociation rates were determined using a bivalent analyte model for curve fitting.

Development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors.

A biosensor-based assay, using a surface plasmon resonance detection system, was developed to detect and isotype anti-adenoviral antibodies in patients dosed with an adenoviral-based gene therapy vector. In the assay, whole, intact virus was immobilized onto the sensor chip surface. Electron microscopy and monoclonal antibody studies provide evidence that the virus remains intact after immobilization. The patients tested had preexisting serum levels of anti-adenoviral antibodies. A classic anamnestic response was observed in patients dosed with the gene-therapy agent. Isotyping experiments indicated that IgG antibodies predominated in serum even at the predose time point. Analysis of ascites fluid samples from some patients indicated detectable levels of IgA in addition to IgG. Results obtained using the biosensor-based assay corresponded to an existing enzyme-linked immunosorbent assay. The assay was easy to perform and the automated instrument reduced the required "hands on" time. In addition to studying the development of anti-adenoviral antibodies, the techniques described may be applied to virus:receptor interaction studies or antiviral drug:virus interaction studies.

The use of field emission scanning electron microscopy to assess recombinant adenovirus stability.

A field emission scanning electron microscopy (FESEM) method was developed to assess the stability of a recombinant adenovirus (rAd). This method was designed to simultaneously sort, count, and size the total number of rAd viral species observed within an image field. To test the method, a preparation of p53 transgene-expressing recombinant adenovirus (rAd/p53) was incubated at 37 degrees C and the viral particles were evaluated by number, structure, and degree of aggregation as a function of time. Transmission electron microscopy (TEM) was also used to obtain ultrastructural detail. In addition, the infectious activity of the incubated rAd/p53 samples was determined using flow cytometry. FESEM image-analysis revealed that incubation at 37 degrees C resulted in a time-dependent decrease in the total number of detectable single rAd/p53 virus particles and an increase in apparent aggregates composed of more than three adenovirus particles. There was also an observed decrease in both the diameter and perimeter of the single rAd/p53 viral particles. TEM further revealed the accumulation of damaged single particles with time at 37 degrees C. The results of this study demonstrate that FESEM, coupled with sophisticated image analysis, may be an important tool in quantifying the distribution of aggregated species and assessing the overall stability of rAd samples.

A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness.

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.