Application of Pharmacokinetic/Pharmacodynamic Modeling to Bridge Mouse Antitumor Efficacy and Monkey Toxicology Data for Determining the Therapeutic Index of an Interleukin-10 Fc Fusion Protein

Pharmacokinetic/pharmacodynamic (PK/PD) modeling was performed to quantitatively integrate preclinical pharmacology and toxicology data for determining the therapeutic index (TI) of an interleukin-10 (IL-10) fragment crystallizable (Fc) fusion protein. Mouse Fc fused with mouse IL-10 (mFc-mIL-10) was studied in mice for antitumor efficacy, and the elevation of interleukin-18 (IL-18) was examined as a PD biomarker. The in vivo mFc-mIL-10 EC50 for the IL-18 induction was estimated to be 2.4 nM, similar to the in vitro receptor binding affinity (Kd) of 3.2 nM. The IL-18 induction was further evaluated in cynomolgus monkeys, where the in vivo induction EC50 by a human IL-10 human Fc-fusion protein (hFc-hIL-10) was 0.08 nM vs. 0.3 nM measured as the in vitro Kd. The extent of the IL-18 induction correlated with mouse antitumor efficacy and was used to connect mouse efficacy to that in monkeys. The PD-based efficacious dose projected in monkeys was comparable to the results obtained using a PK-based method in which mouse efficacious exposure was targeted and corrected for affinity differences between the species. Furthermore, PK/PD relationships were developed for anemia and thrombocytopenia in monkeys treated with hFc-hIL-10, with thrombocytopenia predicted to be dose-limiting toxicity. Using quantitative pharmacology and toxicology information obtained through modeling work in the same species, the TI of hFc-hIL-10 in monkeys was determined to be 2.4 (vs. PD-based efficacy) and 1.2–3 (vs. PK-based efficacy), indicating a narrow safety margin. The model-based approaches were proven valuable to the developability assessment of the IL-10 Fc-fusion protein.

The concentrations of mouse IL-10 mouse Fc-fusion protein (mFc-mIL-10) in mouse samples (10% blood in Rexxip A buffer or serum) were measured using a microfluidic fluorescence immunoassay on a Gyrolab xP Workstation (Gyros AB, Uppsala, Sweden). A biotinylated rat anti-mIL-10 antibody (Southern Biotech, Birmingham, AL) was used as the capture reagent, and a goat anti-mouse immunoglobulin G (IgG) antibody labeled with Alexa Fluor 647 (Southern Biotech, Birmingham, AL) was used as the detection reagent. For diluted blood samples (10% blood in Rexxip A buffer), standard curves and quality control (QC) samples defining the dynamic range of the bioanalytical method were prepared in the 10% mouse blood with Rexxip A buffer and processed in the same fashion as the test samples. For serum samples, they were analyzed at 5fold dilution in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA) and 1M sodium chloride; the corresponding standard and QC samples were prepared in the same way as the serum test samples. Aliquots of diluted test samples, QC samples, standards, and reagents were added to 96-well polymerase chain reaction (PCR) microplates (Thermo Scientific, Cambridge, MA). The Gyrolab workstation transferred samples and reagents from the microplates to each of the microstructures within a Gyrolab Bioaffy compact disc (CD) and spun each CD at the optimized speed and time controlled by the Gyros software to ensure uniform optimal reaction times throughout the integrated assay workflow. All assay steps were automated and controlled by the Gyros control software. The mFc-mIL-10 concentrations in the test samples were quantified by a log-log linear-fit regression model using the SoftMax Pro software (Molecular Devices, Sunnyvale, California). Standard curves and QC samples were evaluated using target acceptance criteria for inaccuracy and imprecision of ± 20% of the nominal concentration to be considered acceptable for assay performance. The lower limit of quantitation (LLOQ) in diluted blood and serum samples were 0.4 and 3 ng/mL (0.004 and 0.03 nM), respectively. The LLOQ in plasma after the conversion of drug concentrations from diluted blood samples using a theoretical dilution factor of 17.36 (see Section 2.5 Data Analysis for details) was 6.9 ng/mL (0.08 nM).

Quantitation of mouse IL-18
Serum mouse IL-18 (mIL-18) assay was developed using Simoa (single molecule array) homebrew 2.0 assay starter kit (Quanterix, Billerica, MA). The Simoa mIL-18 homebrew assay was a 3-step digital immunoassay using the Simoa HD-1 Analyzer. In the 3-step assay, mIL-18 antibody-coated paramagnetic beads were incubated with the samples. mIL-18 present in the samples was then captured by the mIL-18 antibody-coated beads. After washing, a biotinylated IL-18 detector antibody was incubated with the beads. The mIL-18 detector antibody was then bound to the captured mIL-18. Subsequently, streptavidin-β-galactosidase (SBG) was bound to the biotinylated mIL-18 detector antibody, resulting in the enzyme labeling of the captured mIL-18. Following a final wash, the beads were resuspended in a resorufin β-D-galactopyranoside (RGP) substrate solution and transferred to the Simoa Disc. Individual beads were then sealed within microwells in the array. The β-galactosidase hydrolyzed the RGP substrate in the microwell into a fluorescent product that provided the signal for the quantification.
Coupling of mIL-18 capture antibody to the Quanterix magnetic beads utilized carboxyl groups on the beads to conjugate to primary amines on the antibody via a 1-ethyl-3-(-3-dimethyl aminopropyl)carbodiimide hydrochloride (EDC) linker. Beads (200 µL at 1.4 x 10E 9 /mL) were activated by EDC (0.5 mg/mL) for 30 minutes at 4°C. The capture mIL-18 antibody (200 µL at 0.2 mg/mL in the conjugation buffer) was added to the activated beads and incubated for 3 hours at 4°C. The mIL-18 antibody-conjugated beads were washed and blocked with BSA. To prepare for test samples, an aliquot of 5 µL serum sample was diluted into 145 µL of the sample diluent at 30-fold dilutions. The mIL-18 calibration curve was prepared from the mIL-18 ELISA kit (MBL International Corporation, Woburn, MA), in which a stock solution of 2500 pg/mL was made in calibration diluent (from Simoa homebrew kit). The standards ranged from 0 to 250 pg/mL. The mIL-18 assay was run on the Simoa HD-1 Analyzer (Quanterix, Billerica, MA) as a 3-step run. The Simoa mouse IL-18 assay reagents were loaded into the reagent bay in the HD-1 analyzer: 5 mL of bead reagent, 12 mL of detector reagent containing the biotinylated IL-18 antibody at 1:1000 dilution, and 12 mL of SBG Reagent (150 pM) per 96-sample run. The samples, calibrators, and RGP (resorufin-β-D-galactopyranoside) were loaded into the sample bay. The Simoa mIL-18 assays used a 4-parameter curve fit data reduction method (1/y 2 weighted) to generate the calibration curve. The LLOQ of the assay was 0.11 pg/mL. After correcting for sample dilution factors, the LLOQ in serum samples was 3.3 pg/mL.

Quantitation of human IL-10 human Fc-fusion protein
The concentrations of human IL-10 human Fc-fusion protein (hFc-hIL10) in the cynomolgus monkey plasma samples were measured using a chemiluminescence immunoassay platform. Commercial rat anti-hIL-10 (Southern Biotech, Birmingham, AL) was used as the capture reagent. Samples, standards, and QCs were brought up to a final matrix concentration of 10% monkey plasma in PBS with 1% BSA and 0.05% Tween 20 (assay buffer) and loaded in the capture reagent coated, blocked 96-well flat-bottom Nunc MaxiSorp black plate (Thermo Fisher Scientific, Waltham, MA). After overnight incubation at 4°C and wash steps, the detection reagent, biotinlabeled mouse anti-human IgG Fc mAb was added. Followed by another incubation for 2 hours at 4°C and wash steps, NeutrAvidin protein conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA) was added. After final incubation and wash steps, Pico substrate solution (Thermo Fisher Scientific, Waltham, MA) was added to assay plates and read in SpectraMax plate reader at the luminescence mode. The concentrations of hFc-hIL-10 in monkey plasma samples were calculated from luminescence intensity using a log-log linear calibration curve. Calibrators and QCs prepared in monkey plasma were diluted 10-fold in assay buffer and analyzed on each assay plate along with samples to ensure acceptable assay performance. Assay performance was within the acceptable range: % CV of the standards and QC was below 20 %, and QC recovery was within ± 20 % of the nominal values. The LLOQ in monkey plasma samples was 0.5 ng/mL (0.0055 nM).
The Simoa human IL-18 assay kit is a 3-step digital immunoassay using the Simoa HD-1 Analyzer. The 3-step assay principle was the same as that described in the mIL-18 Simoa assay. The cIL-18 standards were prepared from the hIL-18 kit using a stock solution of 1000 pg/mL in the calibration diluent. The standard concentration ranges from 0 to 45 pg/mL. The plasma samples (3 µL) were diluted into 150 µL of the sample diluent and run on the Simoa HD-1 Analyzer using a 3-step run protocol. The Simoa cIL-18 assay uses a 4-parameter curve fit data reduction method (1/y 2 weighted) to generate the calibration curve. The LLOQ of the assay was 0.011 pg/mL. After accounting for the sample dilution, the LLOQ in plasma samples was determined to be 0.56 pg/mL.

Semi-quantitative detection of antidrug antibody in cynomolgus monkeys
Antidrug antibodies (ADA) in monkey plasma samples were detected on a chemiluminescence platform. hFc-hIL-10 and a mouse anti-monkey IgG with horseradish peroxidase (Southern Biotech, Birmingham, AL) were used as the capture and detection reagents respectively in 96-well flat-bottom Nunc MaxiSorp black plate (Thermo Fisher Scientific, Waltham, MA). Samples were analyzed at a 10-fold dilution in assay buffer (PBS with 1% BSA and 0.05% Tween 20) and incubated in the capture reagent coated, blocked assay plate overnight at 4°C. After incubation and wash steps the detection reagent was added. After another incubation for 2 hours at 4°C followed by wash steps, Pico substrate solution (Thermo Fisher Scientific, Waltham, MA) was added and read in SpectraMax plate reader at luminescence mode. The presence of detectable anti-hFc-hIL-10 antibodies in monkey plasma samples was determined by comparing the sample raw signal to the pre-dose raw signal. The samples with a signal higher than the pre-dose signal were considered positive.

Hematology
Peripheral blood smears from the sample collected were prepared and analyzed using the ADVIA 2120i Hematology System (Siemens Healthineers AG, Erlangen, Germany). mFc-mIL-10 concentration: blood (60 µL) was collected serially via submandibular bleeds to obtain serum samples at 4 and 168 hours from tumor-bearing mice in the efficacy study at all dose levels (N = 5 per time point); additionally, blood (60 µL each) was harvested via submandibular bleeds to collect serum samples compositely at 1, 5, 24, 72, 168, 240, and 336 hours compositely from the satellite groups of non-tumor-bearing mice at 0.3 and 3 mg/kg in the same study (N = 3 per time point). All the study samples were stored at ≤-70°C until sample analysis for mFc-mIL-10 concentrations.

Study#1
Single IP dose at 0.1, 0.3, and 1 mg/kg Efficacy data; mFc-mIL-10 concentration: blood (10 µL) was collected compositely via microsampling on the tail at 4, 48, 96, 168, 336, and 504 hours from tumor-bearing mice in the efficacy study at all dose levels (N = 4 or 6 per time point) and diluted into 90 µL Rexxip buffer (Gyros AB, Uppsala, Sweden) for analyses. All the study samples were stored at ≤-70°C until sample analysis for mFc-mIL-10 concentrations.

Study#2
Single IP dose at 0.1, 0.3, and 1 mg/kg Efficacy data; mFc-mIL-10 concentration: blood (10 µL) was harvested serially via microsampling on the tail at 4, 24, 48, 96, and 168 hours from tumor-bearing mice in the efficacy study at all dose levels (N = 4 per time point) and diluted into 90 µL Rexxip buffer for analyses. All the study samples were stored at ≤-70°C until sample analysis for mFc-mIL-10 concentrations.

Study#3
Single IP dose at 0.03, 0.1, and 0.3 mg/kg Efficacy data; mFc-mIL-10 concentration: blood (10 µL) was obtained serially via microsampling on the tail at 4, 24, 48, 96, 168, and 192 (or 216) hours from tumor-bearing mice in the efficacy study at all dose levels (N = 4 per time point) and diluted into 90 µL Rexxip buffer for analyses. All the study samples were stored at ≤-70°C until sample analysis for mFc-mIL-10 concentrations.
All the study samples were stored at ≤-70°C until sample analysis for mFc-mIL-10 concentrations.
cIL-18 concentration: same samples collected for hFc-hIL-10 concentrations were used. Samples were stored at -70°C until analysis for determining cIL-18 levels.
Hematological (hematocrit and platelet) assessment: blood (1 mL) was collected serially from the femoral vein into K2EDTA tubes at predose, 24, 48, 72, 96, and 192  Anti-hFc-hIL-10 antibodies: blood (0.2 mL) was harvested serially from the femoral vein to obtain serum samples at predose and subsequently every week until the 6 th week after the initial dosing. Samples were stored at -70°C until analysis for determining hFc-hIL-10 concentrations.
Note: Submandibular bleeds were performed with the use of a sterile 5mm Goldenrod animal lancet (Braintree Scientific Inc., Braintree, MA). Animals were briefly restrained and an applicable volume of blood was collected from a puncture made proximal of the mandibular bone where the submandibular vein and facial vein converge.     Figure S4. Residual plots obtained from pharmacokinetic/pharmacodynamic modeling of average PK and cIL-18 induction data in cynomolgus monkeys A. PK data (single-dose IV data at 0.005, 0.05, and 0.5 mg/kg; repeat-dose IV data at 0.06 and 0.18 mg/kg Q2W x 3 doses)