In vivo xenograft tumor model

To establish a xenograft tumor model, A549 cells (3×10⁶ in 100 μL PBS; target cells) were mixed with freshly isolated PBMCs (6×10⁷ in 100 μL PBS; effector cells) at the E:T ratio of 20:1 and subcutaneously injected into the right axilla of the nude mice (Day 0). From Day 0 to Day 4, hEND-CD3/BiTE in 100 μL PBS was injected daily into the tail vein at 1, 10, or 100 μg. hENDM-CD3/BiTE; PBS was used as a control. To assess the in vivo specificity of hEND-CD3/BiTE, recombinant human endoglin was added into hEND-CD3/BiTE solution to a final concentration of 100 μg/mL prior to injection into the tail vein. The tumor length (L) and width (W) were measured every five days starting from Day 5 until Day 30, and the tumor volume (V) was calculated as V = L × W²/2.

To evaluate its in vivo safety, hEND-CD3/BiTE was intravenously injected into the mice receiving A549 and PBMCs at 20 mg/mL PBS daily for five consecutive days. Age-matched mice not receiving any injection or treatment were used as controls.