Development of Novel Antibody–Camptothecin Conjugates

ADC pharmacokinetic and physiochemical measurements. A, Size-exclusion chromatography traces for CPT–Lb and CPT–Lc cAC10 ADCs. B, Hydrophobic interaction chromatography. cAC10, cAC10—CPT–Lb, and cAC10-DT were injected onto a Butyl HIC NPR column (2.5 μm, 4.6 mm × 3.5 cm, Tosoh Bioscience, PN 14947) at 25°C and eluted with a 12-minute linear gradient from 0% to 100% B at a flow rate of 0.8 mL/min (Mobile Phase A, 1.5 mol/L ammonium sulfate in 25 mmol/L potassium phosphate, pH 7; Mobile Phase B, 25 mmol/L potassium phosphate, pH 7, 25% isopropanol). C, Kupffer cell uptake assay. Kupffer cells were incubated with AlexaFluor 647-labeled ADCs at a concentration of 0.1 mg/mL in cell culture media for 24 hours. ADC uptake into cells determined by measuring mean fluorescent intensity (MFI), D, Pharmacokinetic profile of h00 mAb (non–cross-reactive), and h00–CPT–Lb in rats. Rats were injected with 1 mg/kg of parental h00 mAb or h00–CPT–Lb ADC. Samples from scheduled blood draws were obtained, and the h00 antibody and h00–CPT–Lb ADC were captured from plasma via a biotin-conjugated murine anti-human light chain kappa mAb and streptavidin-coated beads. The h00 antibody and ADC were quantified via ELISA using an AF647-anti–human kappa detection reagent.

In vivo xenograft studies. A and B (data from separate studies), Activity of cAC10 ADCs based on CPT–Lb, CPT–Lc, and GT drug-linkers in L540cy HD xenograft model. C, Comparison of cAC10–CPT–Lc and DT ADCs in CD30-low L428 HD model. D, Comparison of cAC10–CPT–Lc and cAC10-DT in Karpas 299 (CD30-positive)/Karpas BVR (CD30-negative) bystander killing activity model. All experiments were single-dose studies.

Characterization of CPT drug release and biochemical potency. A, CD30-positive Karpas 299 and L540cy cells were treated with the cAC10–CPT–Lc ADC (100 ng/mL) for 24 hours, followed by extraction of released drug from pelleted cells and supernatant, and drug quantitation by LC-MS/MS. B, Supercoiled DNA-topoisomerase 1 complex trapping with CPT payloads CPT1 and CPT2.