Pharmacokinetics, pharmacodynamics and safety of QGE031 (ligelizumab), a novel high affinity anti-IgE antibody, in atopic subjects

Jonathan P. Arm, Ivan Bottoli, Andrej Skerjanec, David Floch, Andrea Groenewegen, Suzanne Maahs, Charles E. Owen, Ieuan Jones and Philip J. Lowe

Supplementary Materials

Methods

Preclinical pharmacology

Equilibrium constant:

Each monoclonal antibody (omalizumab and QGE031) was diluted to 5 μg/mL in 10 mM sodium acetate pH 4.5 and immobilized at a flow rate of 5 μL/min on a CM5 biosensor chip by standard amine coupling with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride, N-hydroxy-succinimide and ethanolamine-hydrochloride (Biacore, Uppsala, Sweden). All monoclonal antibodies were tested by surface plasmon resonance on a Biacore 3000 optical biosensor (Biacore, Uppsala, Sweden) at 22°C against polyclonal human IgE analytes. Human IgE analytes were injected onto the chip at five concentrations (0.625, 1.25, 2.5, 5 and 10 nM) at a flow rate of 30 μL/min for 5 min and allowed to dissociate for 30 min. The chip surface was regenerated at 100 μL/min with 100 μL of 100 mM HCl. The ligand immobilization level was set at 100 RU. Sensorgrams were plotted with Biacore 300 control software (version 4.1).

Inhibition of IgE binding to cell-surface FcεRI and the immobilized α-subunit of FcεRI:

A predetermined amount of biotinylated human IgE was mixed with serially diluted monoclonal antibodies (QGE031 and omalizumab) and added to RBL-2H3 (a gift from Dr F. Kricek, Novartis Dermatology, Vienna, Austria) cells expressing the α-subunit of the human FcεRI receptor on their cell surface. After washing, bound IgE was detected using FITC-conjugated streptavidin and mean fluorescence intensity was determined.

Serially diluted monoclonal antibodies (QGE031 and omalizumab) were loaded onto an ELISA plate coated with 1 μg/mL recombinant soluble human FcεRIα receptor. IgE (200ng/mL) was added to the plate and allowed to bind for 1 h at room temperature, thus allowing IgE the opportunity to bind either with the monoclonal antibodies or the receptor. The plates were washed, and IgE bound to the receptor was determined by a horseradish peroxidase-labeled murine anti-human IgE monoclonal antibody, E10-10 (Tanox Inc, Houston, TX, USA).

Mast cell degranulation and activation assays:

Human cord blood-derived mast cells at a concentration of 1 x 105 cells/80 μL in culture medium supplemented with 25 ng/mL recombinant IL-4 were added to wells of 96-well V-bottom plates. Ten μL aliquots of serial dilutions of monoclonal anti-IgE antibodies (omalizumab, QGE031) or an isotype-matched control IgG1 monoclonal antibody were added to the cells, followed by JW8 3-nitro-4-hydroxy-iodophenyl acetic acid (NIP)-specific IgE to a concentration of 1 nM and incubated overnight. Cells were then spun, washed once with 200 μL of culture medium and resuspended in 90 μL of the assay medium. Ten μL of the stimulus (100 ng/mL of NIP-BSA conjugated hapten antigen) or 10 μL of assay medium was added and the plates were incubated for 2 h at 37°C. The plates were then spun. Cell pellets from three replicates were combined and stained with anti-CD63-PE monoclonal antibody (Immunotech) to determine the expression of CD63 at the cell surface by flow cytometry. β-hexosaminidase release was measured as described previously [1].

Binding activity of QGE031 across mammalian species:

Binding activity of QGE031 against IgE from different mammalian species was investigated using ELISA measured by absorbance at 450 nm for rat (Zymed), cat (Heska), dog (Heska), and human (Biodesign), while QGE031 binding to cynomolgus non-human primate was assessed by Biacore 3000.

Immulon II U-bottom 96-well plates were coated with 50 μL/well of 1μg/mL of polyclonal human IgE, mouse IgE, cat IgE, rat IgE and dog IgE overnight at 4°C. Coating solutions were removed and the plate blocked with 3% bovine serum albumin in phosphate buffered saline (200 μL/well) for 1 h at room temperature. After washing the plates five times, 50 μL/well of primary antibodies or control antibody were added and incubated for 1 h at room temperature, followed by a further five washes. Goat anti-human IgG Fc-horseradish peroxidase (1:2000) was added, incubated for 1 h at room temperature and the plates were washed a further five times. Fifty μL of TMB substrate (3,3/, 5, 5/-tetramethyl-benzidine liquid substrate system) was added to develop the reaction. Fifty μL of 0.2 M sulfuric acid was added to stop the reaction, and the plate was read in a ELISA plate reader at an optical density 450/570 nm.

Monoclonal antibodies (omalizumab and QGE031, both at 5 μg/mL) in 10 mM sodium acetate (pH 4.5) were immobilized at a flow rate of 5 μL/min on a CM5 biosensor chip by standard amine coupling with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride, N-hydroxy-succinimide and ethanolamine hydrochloride. Different concentrations of the IgE analytes from three species (human, cynomolgus monkey and rhesus monkey) were injected onto the chip at a flow rate of 30 μL/min over 3–5 min and allowed to dissociate for 30 min. The chip surface was regenerated at 100 μL/min with 100 μL 100 nM hydrochloric acid. A reference flow cell (phosphate buffered saline) was also prepared. A blank (phosphate buffered saline) was injected under the same conditions as the IgE analytes. Biacore software was used to determine the cross-species binding.

Methods

Clinical trials

Dose selection:

The intravenous study was the first-in-human study and was therefore conducted without prior information of the pharmacokinetics or pharmacodynamics of QGE031 in humans. The starting dose of 0.1 mg/kg was chosen based on several factors. Firstly, the 12-fold difference in dissociation constant from non-human primates where doses up to 120 mg/kg were given allowed for a possible starting dose of 10 mg/kg. A standard safety factor of 10-fold when extrapolating from animals to man reduced the starting dose to 1 mg/kg. Finally, when comparing the in vitro suppression of free IgE by QGE031 with that of an anti-IgE of similar affinity, HAE-1 [2], the dose of QGE031 that would provide a similar suppression was estimated to be 10-fold lower, allowing a starting dose of 0.1 mg/kg. As this was a first-in-human study, the first two subjects in Cohort 1 were dosed on consecutive days in an open-label manner. The remaining subjects in Cohort 1 were dosed in a double-blind manner. For subsequent Cohorts (2–6), subjects were dosed in a double-blind manner with at least 2 h separating the dosing of each subject.

The highest dose of QGE031 for the subcutaneous study was based upon model-based pharmacokinetics simulations that predicted an adequate exposure margin compared with exposure to non-human primates in preclinical safety studies

Institutional Review Boards:

There was one Institutional Review Board (IRB) for the intravenous study (Aspire IRB, CA, USA) and three IRBs for the subcutaneous study (Aspire IRB, CA, USA; Quorum Review Inc, WA, USA and Alpha IRB, CA, USA).

Protocol amendments:

Intravenous trial

The study protocol was amended five times, and most amendments included administrative and personnel changes. None of the amendments were considered to have affected the interpretation of study results as they occurred prior to unblinding. Of the five amendments, the most significant was the introduction of the placebo expansion group (Cohort 6a); Amendment 5, 13 November 2009.

Subcutaneous trial

The protocol was amended once after a planned interim analysis that revealed no urticarial events in at least 36 subjects who had received four doses of QGE031 (Amendment 1, 02 February 2011). The amendment included an additional cohort of subjects who were to receive a higher dose of QGE031 (Cohort 6) to further explore the safety, tolerability, pharmacokinetics and pharmacodynamics of QGE031 to facilitate dose selection for subsequent Phase II studies. The amendment also modified the visit schedule of subjects who received 4-weekly doses of omalizumab. The original version of the protocol assumed biweekly dosing of omalizumab. However, several subjects were dosed monthly in accordance with the established dosing table [3] and the modified visit schedule streamlined visits and assessments on non-dosing days. The amendment allowed inclusion of subjects with atopic dermatitis to facilitate recruitment of subjects with IgE > 700 IU/mL.

Pharmacokinetic measurement of QGE031 in serum:

One hundred microliters of 2 μg/mL biotin conjugated mouse-anti-human-IgE antibody (BD

Pharmingen No. 555858, Batch 07164, 0.5 mg/mL) was coated onto each well of streptavidin-precoated 96-well microplates (F96, No. 436014, Nunc). The plates were placed on a microplate shaker (Titramax 1000, Heidolph, Merck) and incubated in the dark at room temperature for 1 h. The plates were then washed three times with 300 μL/well of wash buffer (Tris Buffered Saline with Tween-20® pH 8.0, No. T-9039, Sigma). Ten microliters of standard samples, unknowns and quality controls were added to 475 μL of sample dilution buffer (Low Cross Buffer, Pierce), together with 25 μL of 100 μg/mL of human-IgE solution (Myeloma Biodesign, A50796H, batch 1E14405, 1.199 mg/mL). After shaking for 30 min at room temperature, 100 μL was added to each well. The plates were placed on the microplate shaker and incubated for 2 h in the dark at room temperature.

The plates were washed four times with wash buffer to remove any unbound substance. One hundred microliters of an alkaline phosphatase-linked mouse monoclonal antibody specific for human IgG (1:10000 dilution of 1.8 mg/mL, No. A2064, Lot 077K4793, Clone CG-5, Sigma) was added to bind to the captured QGE031, and the plate was again incubated (shaking) for 1 h in the dark at room temperature. After three additional washing steps to remove unbound reagent, 100 μL of substrate solution (No. UP664791, Uptima) was added and incubated for 50 min (shaking) in the dark at room temperature. The plates were read using a Vmax Microplate reader (Molecular Devices) at 405 nm using SoftMax Pro (version 5.0.1) software.

Pharmacodynamic assessments - total IgE:

One hundred microliters of 0.5 μg/mL biotin conjugated goat-anti-human-IgE antibody (Ref. A80-108A, Bethyl) was coated onto each well of streptavidin-precoated 96-well microplates (F96, No. 436014, Nunc) and placed on a microplate shaker (Titramax 1000, Heidolph, Merck) and incubated in the dark at room temperature for 1 h. The plates were then washed three times with 300 μL/well of wash buffer (Tris Buffered Saline with Tween-20® pH 8.0, No. T-9039, Sigma). One hundred microliters of standard samples, unknowns and quality controls were added to 100 μL of a solution of QGE031 (300 μg/mL) and shaken for 30 min at room temperature to complex all the IgE in the sample. The complexes were then further diluted 1:10 with Low Cross Buffer (Pierce) and 100 μL added to each well followed by incubation (shaking) for 2 h at room temperature in order to capture the complex with the coated goat-anti-IgE antibody.

The plates were washed three times with wash buffer to remove any unbound substance. One hundred microliters of an alkaline phosphatase-linked mouse monoclonal antibody specific for human IgG (1:10000 dilution of 1.8 mg/mL, No. A2064, Lot 077K4793, Clone CG-5, Sigma) was added to bind to the captured QGE031 and the plate was again incubated (shaking) for 1 h in the dark at room temperature. After three additional washing steps to remove unbound reagent, 150 μL of substrate solution (No. UP664791, Uptima) was added and incubated for 60 min (shaking) in the dark at 37°C. Optical density was measured using a Vmax microplate reader (Molecular Devices) at 405 nm and using SoftMax Pro (version 5.0.1) software.

Pharmacodynamic assessments - free IgE:

One hundred microliters of IgE high-affinity receptor (FcεRI conjugated, 1.5 μg/mL, Novartis Pharma AG) was added to each well of 96-wells microplates (MaxiSorp plates, No. 439454, Nunc) and incubated over night at 4°C. The plates were then washed four times with 300 μL/well of wash buffer (Tris Buffered Saline with Tween-20® pH 8.0, No. T-9039, Sigma). Three hundred microliters of Superblock Blocking Buffer (No. 37535, Pierce) was added to each well and the plate was placed on a microplate shaker (Titramax 1000, Heidolph, Merck) and incubated for 20 min at room temperature. The plates were then washed a further four times with wash buffer. Each standard sample, unknowns and quality controls were diluted 1:5 (Low Cross Buffer, Pierce) and 100 μL of diluted sample was added to each well and incubated for 2 h (shaking) at room temperature in the dark.

The plates were washed four times with wash buffer to remove any unbound substance. One hundred microliters of an alkaline phosphatase-linked mouse monoclonal antibody specific for human IgE (No. 555859, BD) was added and the plate was again incubated (shaking) for 1 h in the dark at room temperature. After six washing steps to remove unbound reagent, 50 μL of substrate solution NADPH was added to each well and incubated for 15 min (shaking) at room temperature. Finally, 50 μL of Amplifier solution (No. 19589-019, Invitrogen) was added to each well and then incubated (shaking) for 20 min at room temperature. Fifty microliters of Stop solution (Sulfuric acid) was added to terminate the reaction. Optical density was measured using a VersaMax microplate reader (Molecular Devices) at 495 nm using SoftMax Pro (Version 5.0.1) software.

Pharmacodynamic assessments: fluorescence-activated cell sorting analysis:

Antibodies and reagents

Basophil selection: Mouse anti-human Lineage cocktail-FITC, monoclonal antibody cocktail-FITC (BD 340546), Mouse anti-Human CD203c-PerCp-Cy5.5 monoclonal antibody (BioLegend 324608). For the expression levels of FcεRI and IgE: Mouse monoclonal anti- Human FcεRI-APC (eBioscience 17-5699), mouse monoclonal anti-human IgE-PE (BioLegend 325506).

Protocol

At the clinical site antibody mixes were added to 200 µL aliquots of whole blood and incubated for 30 min at room temperature. Lyse/fix buffer (BD 349202) was then added and the samples frozen at -80°C and sent to the fluorescence-activated cell sorting analysis laboratory (Cellular Biomarker Group, Novartis Institutes for Biomedical Research, Basel, Switzerland). After thawing and antibody-staining with the antibodies mentioned above, 2000 basophils were acquired for each sample by flow cytometry performed on a fluorescence-activated cell sorting Canto II cytometer. Molecules of equivalent soluble fluorochrome values were calculated using Quantum molecules of equivalent soluble fluorochrome beads (Bangs Laboratories Inc.)

Pharmacokinetic and pharmacodynamic model of binding to and capture of IgE by QGE031 and omalizumab:

The pharmacokinetic profile of QGE031 and several pharmacodynamic parameters including total IgE, basophil FceRI and basophil surface IgE were analyzed using an adaptation of the previously published omalizumab PK-IgE binding model [4-6]. The published model was updated to include: i) two compartments instead of one to allow the analysis of the intravenous pharmacokinetics of QGE031; ii) basophil FcεRI and surface IgE as indirect responses to free IgE suppression; iii) both QGE031 and omalizumab with the assumption that the only difference between the compounds was their ability to bind IgE given that they are both IgG1 monoclonal antibodies. Model parameters for drug clearances and volumes of distribution, binding of QGE031 or omalizumab to and turnover of IgE, the effect of free IgE on basophil FceRI and surface IgE were estimated to get the mathematical equations to fit the clinical time course data for the pharmacokinetic and pharmacodynamic assessments. As in prior publications, the NONMEM® software (Icon plc, Dublin, Ireland) was used for the model based analysis.