(A) IHC of pre-clinical, cynomolgus lung tissue 24 hr after intravenous single-dose administration of high-affinity FOLR1-TCB (FOLR1(Hi), 10 µg/kg), demonstrating leukocytic infiltration (dark purple cells) and inflammation. (B) Expression of FOLR1 protein in healthy cynomolgus lung tissue stained with antibody targeting FOLR1. (C, F) Isotype controls of FOLR1 staining in healthy cynomolgus and human lung tissues, respectively. (D) High FOLR1 expression displayed in human ovarian carcinoma HeLa cell line for comparison to (E) histopathological staining of primary healthy human lung tissue for FOLR1. (G) Schematic of Alveolus Lung-Chip to model human FOLR1 on-target toxicities. Alveolus Lung-Chip design is composed of a top microfluidic channel (Waldman et al., 2020) seeded with primary adult human alveolar cells (Yang, 2015) cultured to maturity with air–liquid interface (ALI). The top, epithelial channel is separated with a flexible, porous membrane (Hodi et al., 2010) from a bottom, vascular channel seeded with primary lung microvascular cells (Schadendorf et al., 2015; Wolchok et al., 2017). Mechanical stretching is applied via pneumatic actuation of parallel vacuum channels (Gong et al., 2018). (H) RNAseq expression levels of FOLR1 gene in cultured alveolar epithelial cells on day 0 (before seeding), 5, or 10 after seeding and differentiation on the Alveolus Lung-Chip (n=3, ± SEM). (I) Representative immunofluorescent staining of chip epithelium (Nuclei, blue) at day 10 of culture expressing the tight junction marker E-cadherin (green) and FOLR1 target antigen (red). Images taken at 40× magnification. (J) Estimation of surface FOLR1 binding site expression via flow cytometry of harvested chip epithelial cells at days 0 (before seeding), 5, and 10 (n=2–4, ± SEM).