Bi-specific single change antibody fragments (bi-scFv) represent an emerging class of biotherapeutics. We recently developed a fully human bi-scFv (EGFRvIII:CD3 bi-scFv) with the goal of redirecting CD3-expressiong T cells to recognize and destroy malignant, EGFR-expressing glioma. In mice, we showed that EGFRvIII:CD3 bi-scFv effectively treats orthotopic patient-derived malignant glioma and syngeneic glioblastoma. Here, we sought to develop a targeted assay for pharmacokinetic (PK) analysis of EGFRvIII:CD3 bi-scFv, a necessary step in the drug development process. Using microflow liquid chromatography coupled to high resolution parallel reaction monitoring mass spectrometry, and data analysis in Skyline, we developed a bottom-up proteomic assay for quantification of EGFRvIII:CD3 bi-scFv in both plasma and whole blood. Importantly, a protein calibrator, along with stable isotope-labeled EGFRvIII:CD3 bi-scFv protein, were used for absolute quantification. A PK analysis in a CD3 humanized mouse revealed that EGFRvIII:CD3 bi-scFv in plasma and whole blood has an initial half-life of ~8 minutes and a terminal half-life of ~2.5 hours. Our results establish a sensitive, high-throughput assay for direct quantification of EGFRvIII:CD3 bi-scFv without the need for immunoaffinity enrichment. Moreover, these half-life measurements will guide drug optimization and dosing regimens in future pre-clinical and Phase 0/I studies of EGFRvIII:CD3 bi-scFv

Whole blood and plasma samples were spiked with a stable isotope-labeled protein followed by denaturation and reduction with sodium deoxycholate and DTT. Samples were trypsinized with 1:10 TPCK trypsin for 4 h followed by acid precipitation of SDC. An external calibration curve was made by addition of light protein standard to non-dosed plasma or whole blood samples. 50 ug of plasma or 150 ug of whole blood digests were analyzed by parallel reaction monitoring using a Waters ACQUITY LC interfaced to a Thermo Q-Exactive HF-X via a heated electrospray ionization (HESI-II) source, and data was analyzed in Skyline.

PK studies used transgenic mice with heterologous expression human CD3ε (hCD3ε-/+) on a C57B/6 background (tgε600, Jackson Laboratory, strain number 20456 crossed with wild-type C57/BL6). Mice (16 weeks old; 23.5 ± 1.6 (s.d.) g) were administered 100 µg of 12C14N EGFRvIII:CD3 bi-scFv (100 µl of 1 mg/mL drug) via tail vein injection. At each designated time point, blood was collected from n=5 mice via retro-orbital bleeds using heparinized microhematocrit capillary tubes. Whole blood samples were immediately frozen, and plasma samples were isolated by centrifugation of whole blood at 6000 xg for 15 min at 4 °C followed by collection of the cell-free supernatant.