KG207-H, a fusion protein comprising humanized BBB carrier FC5 VHH fused to N-terminus of human IgG1 Fc fragment and fused to Aß-binding peptide (ABP) at its C-terminus [FC5(H3)-hFc-ABP], was designed and expressed in CHO cells. The expressed protein was purified on MabSelect Sure affinity columns and further purified by cation exchange (CEX) chromatography.
FC5 Binding to Dog Endothelial Cells In Vitro
The cross species binding of FC5-hFc in endothelial cells was assessed by mirrorball® high sensitivity microplate cytometry (SPT Labtech Ltd., Melbourn, UK) using protocols published previously (27). Immortalized human (HCMEC/D3/HBEC-D3, Dr. Pierre Olivier Couraud, Cochin Institute Université Paris Descartes INSERM, Ref 28), rat microvascular brain endothelial cells (sv-ARBEC) and Canine Aortic Endothelial Cells (CnAOEC, Cell Applications Inc., San Diego, CA, USA) were cultured until confluent at 37°C in a humidified 5% CO2 atmosphere in T-75 flasks coated with 100 μg/ml rat tail collagen type 1 (BD Canada, Mississauga, ON, Canada).
SV-ARBEC cells were grown in M199 medium supplemented with peptone, D-glucose, BME Amino Acids, BME vitamins, antibiotic/antimycotic and fetal bovine serum (ThermoFisher, Mississauga, ON, Canada). HBEC-D3 cells were cultured in EBM2 basal media (Lonza, Walkersville, MD, USA CC3156) supplemented with one quarter of a SingleQuot kit (Lonza CC 4176) and 2% fetal bovine serum. CnAOEC were cultured in Canine EC Growth Media All in One (Cell Applications, Inc). The cells were dissociated with Trypsin/EDTA (ThermoFisher) and washed in complete M199 serum media. Cell number was calculated using a Bio-Rad TC20 automated cell counter with Trypan Blue dye to assess viability, diluted to 8.0 × 104 cells/ml in appropriate growing media. 50 μl of each cell suspension was added into a Nunc™ MicroWell 96-well optical bottom assay plate (ThermoFisher) and incubated for 24 h to allow cell attachment.
For binding studies, the adherent cells were incubated with 50 μL test antibodies diluted to a final concentration of 1.0 μM in phenol red free M199/25% PBS buffer for 1 hr. at 37°C. The cells were washed in phenol red free M199 assay buffer. Secondary detection reagent AF488-conjugated AffiniPure Donkey Anti-human IgG Fc γ fragment specific antibody (1500 ng/ml Jackson Immunoresearch West Grove, PA, USA) was diluted in assay buffer then added to each well for 45 min at 37°C. The cells were washed with phenol red free M199 then stained with 0.5 μM DRAQ5™ (Cell Signaling Technology, Danvers, MA, USA) for 10 min at room temperature. The data was acquired on a mirrorball® microplate cytometer (STP Labtech Ltd., Melbourn, UK) and analyzed using Cellista software (STP Labtech Ltd) and GraphPad Prism.
Male Wistar rats aged 4-6 weeks and weighing 190-230 g were purchased from Charles River (Montreal, Canada) and allowed to acclimatize to the facility for a period of at least 5 days prior to any procedure. Animals were individually housed in polypropylene cages in a 12 h light/12 h dark cycle with free access to food and water. All animal studies were approved by NRC’s Human Health Therapeutics (HHT) Research Centre Animal Care Committee and were in accordance with Canadian Council on Animal Care (CCAC) guidelines.
In Vivo PK Experiments
Rats were injected with 15 mg/kg or 30 mg/kg KG207-H via lateral tail vein (n = 4 rats per dose). Blood was collected via the tail vein at 0.5, 1, 2, 4, 8, 24, 48, 72 and 168 hours after injection (for the 15 mg/kg dose) and 0.5, 1, 2, 6, 24, 48, 72, 96, 168, 240 and 336 hours after injection (for the 30 mg/kg dose). CSF was collected via cisterna magna puncture at 4, 8, 24, 48, 72, and 168 hours (for the 15 mg/kg dose) and 6, 24, 48, 72 and 168 hours (for the 30 mg/kg dose) after injection as described below.
Serial CSF Collection
Rats were anesthetized with 4% isoflurane and placed in a stereotaxic frame with the head tilted downward at a 45°angle. After removing the fur and cleaning the surgical area, a midline incision was made, beginning at the occipital crest and extending caudally ~2 cm. The neck muscles were retracted to access the atlanto-occipital membrane. The dura mater overlying the cisterna magna was exposed, and a 27G butterfly needle was inserted 1 mm into cisterna magna at a sharp angle to the dura. CSF was slowly aspirated using a 1 ml syringe attached to the needle with a short piece of tubing (~15-20 μl at each collection time point). Samples were then placed in glass sample vials and frozen on dry ice. The skin was closed with sutures. The procedure was repeated up to 5 times per rat at various post-injection intervals.
Blood samples were collected from the tail vein by a 27G needle puncture and transferred to serum separator tubes (BD) and kept at room temperature for 30 min and then centrifuged at 1000 g for 10 min. The serum was transferred to sample (glass) vials and frozen on dry ice immediately.
Beagle Dog Study
This protocol was developed in accordance with principles of the Animals for Research Act of Ontario and the guidelines of Canadian Council on Animal Care (CCAC) and reviewed and approved by InterVivo Solutions’ (Ontario, Canada) Animal Care Committee (ACC) and by NRC’s ACC.
Sixteen healthy Beagle dogs (5 males and 11 females, ranging from 7 to 13 years) in good health were included in this parallel group, non-blinded, study. Subjects were placed into four groups matched with respect to age, body weight and gender to the extent possible. Treatment groups did not differ statistically, and groups were randomly assigned to a treatment condition (PBS or KG207-H at 5, 20 or 50 mg/kg body weight) by the drawing of lot.
Subjects received the control or test articles IV via a cephalic catheter at a rate of approximately 8-10 seconds/mL for 5-7 minutes followed by 1-3 mL of a sterile saline flush. Time 0 represented the time the entire dose and saline flush were administered.
Blood samples, approximately 3 ml, were collected via jugular venipuncture prior to dosing and at 0.25, 0.5, 1, 2, 4, and 8 hours (±3 minutes up to the 1-hour collection, ±15 minutes for remaining collections) and at 1, 2, 3, 4, 7, 8, 9, and 14 days (±1 hour) following the drug injection. Samples were transferred into a serum separator blood collection tube (SST) and allowed to clot at room temperature. The tubes were centrifuged at 2800-3200 rpm for 10 minutes at room temperature. For each sample, serum was separated equally into four cryovials.
Cerebrospinal fluid (CSF) was collected prior to dosing and at 2, 4, and 8 hours (±10 minutes), and 1, 2, 3, 4, 7, 8 and 14 days (±1 hour), following injection. Samples were obtained under short term sedation from the cisterna magna using sterile technique. Dogs were anesthetized to effect with propofol (8 mg/kg, IV) and maintained on 0.5-2% isoflurane with oxygen. Between 0.5-1.0 ml of CSF were collected at each time point. CSF were placed on wet ice until being processed by centrifugation at 3000 rpm for 2 minutes at 2-8°C in order to remove potential red blood cell contamination. Once centrifuged, CSF samples were separated into 4 equal aliquots and stored at −80°C (±5°C) until further use.
Drug Level Measurement in Serum/CSF Samples by Multiplexed Mass Spectrometry Analysis
FC5(H3)-hFc-ABP (KG207-H) fusion protein levels in the serum and CSF and Aβ levels in the CSF were quantified using targeted multiplexed mass spectrometry as previously described (11). Briefly, plasma and CSF samples containing KG207-H were reduced, alkylated, and trypsin digested using the previously described protocol (11, 29). For absolute quantification, calibration curves were prepared by spiking KG207-H in the appropriate matrix from naïve animals (either dog or rat plasma at 0 – 10 nmol/mL, or dog or rat CSF at 0 –20 nM) and similarly digested. All digests were analyzed on nanoAcquity UPLC (Waters, Milford, MA) coupled to ESI LTQ XL ETD mass spectrometer (ThermoFisher, Waltham, MA) in selected reaction monitoring (SRM) mode as previously described (17). The SRM method included multiplexed quantification of three peptides unique to KG207-H (FC5 ITWGGDNTFYSNSVK; hFc TTPPVLDSDGSFFLYSK; ABP + linker SLSLSPGTGGGGSGGGGSGTFGTGGASAQASLASK), one host Aβ peptide (LVFFAEDVGSNK), and one host albumin peptide (SLHTLFGDK). Isotopically heavy versions of the FC5, hFc and Aβ peptides, containing heavy C-terminus K (+8 Da), were also synthesized (New England Peptide LLC, Gardner, MA) and included as internal standards for quantification as previously described (11, 29). Raw data extraction and analysis was performed using Skyline software version 3.7 (https://skyline.ms). Lower limits of quantifications were calculated to be 0.02 nmol/mL in serum and 0.1 nM in CSF background. The albumin peptide was used to identify and exclude serum-contaminated CSF samples (11). Total Aβ levels are reported relative to the mean levels in PBS-injected animals (dog) or relative to maximum measured value (rat). During the preparation of samples for multiplexed SRM method analyses, protein samples were denatured and trypsinized to generate peptide fragments. In this process, any protein:protein complex, such as ABP:Aβ in this case, will be disrupted and thus overcomes potential interference in Aβ measurement. Thus, the described mass spectroscopy analytical method has an advantage over conventional ELISA wherein Aβ complex with other proteins might interfere with its accurate measurement unless the complex is disrupted.
Drug Level Measurement in Dog Serum Samples by ELISA
KG207-H levels in dog serum samples were quantified using a sandwich ELISA with FC5 capture and ABP detection. MaxiSorp ELISA plates were coated with anti-FC5 antibody (developed in-house, 1.5 μg/ml) in 20 mM Tris-HCl pH 8.0 at 4°C overnight, then blocked in 1% BSA in TBST. Appropriately diluted serum samples (in TBST) were overlaid on the plates for 1 hour. After rinsing in TBST, the wells were incubated with anti-ABP antibody (developed in-house; 1:5000 for 60 minutes at room temperature), rinsed again, and incubated with HRP-labeled anti-rabbit antibody (Jackson ImmunoResearch; 1:20,000) for 90 minutes at room temperature. Drug levels were quantified using a standard curve established by spiking TBST with KG207-H. The large number of dog serum samples necessitated use of more time- and cost-effective ELISA assay. Analysis of a subset of dog serum samples by both mass spectrometry and ELISA yielded a good correlation between KG207 measurements obtained using the two methods. Mass spectrometry was used for CSF samples since it allowed simultaneous measurements of KG207 and Aβ.
Serum and brain concentration-time profiles were analyzed using WinNonlin software (Version 8.3, Pharsight Corporation, Mountain View, CA, USA). Serum concentration-time data for KG207-H were analyzed using naive pooled data with two-compartment model, IV administration (bolus for rat, short infusion for dog), first-order elimination, and macro-rate constants to estimate the following pharmacokinetic parameters: Volume of distribution of the central compartment (V1) and of the peripheral compartment (V2), Clearance (CL), Inter-compartmental Clearance (CLD) and overall elimination half-life (t1/2β). Overall, the goodness of fit was based upon the predicted estimate and percent coefficient of variation (% CV) for primary and secondary parameters, as well as inspection of residual plots between observed and predicted concentration-time data.
A non-compartmental approach consistent with IV administration and linear/log trapezoidal method was employed to estimate the area under the curve (AUC) of the serum concentration vs time and CSF concentration vs time. For CSF, the AUC from the start of dose administration to the last observed quantifiable measurement (AUC0-168) was estimated. Estimation of average concentration ratios for AUC0-168 is reported as (AUCCSF, 0-168 /AUCSerum, 0-168) × 100.