Abstract Rationale In the therapeutic bleomycin model of pulmonary fibrosis, treatment with ORJ-001, a β1 integrin agonist peptide, reduced established lung fibrosis and repaired the alveolar epithelium. ORJ-001 binds with high selectivity to the primed configuration of β1 integrin in damaged tissues following activation by cytokines and growth factors, with negligible binding to normal tissues. Tenascin-C (TN-C), an extracellular matrix protein expressed during wound healing, correlates to changes in FVC in IPF patients. This study aimed to characterize ORJ-001 in vivo target engagement and the suitability of tenascin-C as a biomarker for ORJ-001. Methods For both target engagement and TN-C studies, mice received bleomycin (15mg/kg) or saline intra-peritoneally (IP) twice a week for 28 days to induce fibrosis. To demonstrate in vivo target engagement, ORJ-001 was labeled with the fluorescent dye Cy5.5 and administered to mice in a single subcutaneous (SC) injection of 60mg/kg on Day 28. Fluorescence images were acquired using IVIS Lumina III in vivo system and then organs were removed and processed for immunofluorescence staining at 30 minutes, 1 h, 8 h, 1 day, 3 days and 7 days post-injection. Tissue sections were stained for β1 integrin, incubated with Alexa Fluor 488-conjugated secondary antibody and counterstained with DAPI. To determine ORJ-001’s effect on TN-C levels, mice were treated with saline or 60mg/kg of ORJ-001 SC on Days 28, 35, 42 and 49 and sacrificed on Day 56. TN-C plasma levels were measured using ELISA. Results Whole-body fluorescence was seen up to 24 h, then declined by Days 3–7. Ex vivo imaging indicated early distribution to liver, intestine, kidney, and lung (30 min–8 h), shifting to predominantly lung signal from Days 1 - 7. ORJ-001 colocalized with β1integrin in fibrotic lungs from Days 1–7. Plasma Tenascin-C levels were elevated in the bleomycin-induced group treated with vehicle (NT group; 1024±92 ng/mL) compared with normal controls at baseline (492±49 ng/mL). Treatment with ORJ-001 induced a gradual decline of TN-C reaching 529±18 ng/mL at 4 weeks of treatment, which was close to the control animals (456±29 ng/mL), while TN-C level remained unchanged (1081±75 ng/mL) in the NT group. Conclusions Binding of ORJ-001 to fibrotic tissue persisted up to 7 days after a single SC injection. ORJ-001 lowered TN-C plasma levels, supporting the use of TN-C as a biomarker of ORJ-001’s response in future studies in IPF patients. This abstract is funded by: Oorja Bio, NIBEC
Pena et al. (Fri,) studied this question.