Next, we further increased the dose to 6 mg/kg, 4XQ1W and found that F12-MMAE almost eradicated tumors (complete remission, CR; %TGI = 98.3%) : For evaluation of in vivo ADCC effects of IgG1 F12, PC-3-PSCA cells (5 × 106 cells/mouse) in 200 ul of 1:1 PBS/Matrigel (BD Biosciences, Cat# 354234) were injected subcutaneously (s.c.) into the right flank of the Balb/c scid mice. When tumor volume reached 100 mm3, mice were divided randomly into IgG1 F12 treatment or vehicle group and i.p. administered four times total with a four-day interval. For evaluation of the ADC (F12-MMAE or Ab1-MMAE) efficacy in vivo, PC-3-PSCA cells (5 × 106 cells/mouse, 5 mice per group) in 200 ul of 1:1 PBS/Matrigel were injected s.c. into the right flank of the NSG mice. Mice were randomly divided into the different treatment groups when tumors reached 100 mm3. We conducted ADC treatment studies at three different dosing regimens, comparing PSCA-ADC with the isotype control Ab1-ADC. The regimens design were as follows: dose 1): 1 mg/kg and 3 mg/kg, administered twice weekly (2XQ1W); dose 2): 6 mg/kg, administered twice weekly (2XQ1W), followed by a single injection of 3 mg/kg when tumors regrew to 140 mm3; dose 3): 6 mg/kg, administered four times weekly (4XQ1W). All ADCs were i.p. administered. Tumor dimensions and mouse weight were measured periodically with a slide caliper and scale. Tumor volume was calculated by the formula: V = 0.5×length × (width)2. Tumor growth inhibition was calculated by the formula: %TGI = [1- (mean tumor volume (MTV)ADC treated/MTVcontrol)] × 100. Mice were euthanized when the tumor volume exceeded 1000 mm3 or if the animals showed any signs of suffering. At the end of the experiment time point, tumors were isolated and weighed. In the dose 2 experiment, one week after two 6 mg/kg ADC treatments, ~50 ul blood were collected to the purple-topped K+/EDTA tubes, and subjected to complete blood counts using the Abaxis HM5 machine from In vivo Imaging Facilities in Hillman Cancer Center of the University of Pittsburgh. In the dose 3 experiment (6 mg/kg, 4×Q1W), mouse spleen, kidney, heart, lung, and liver were collected and fixed in formalin. The H&E staining were completed by the Biospecimen Core of the University of Pittsburgh.

The PC-3 or PC-3-PSCA cells (2.5 × 104 cells) were pre-seeded in 96-well plates overnight. The cells were then incubated with serially diluted ADC, or naked IgG1 antibody or linker-payload combination compound for 4-5 days at 37°C, 8% CO2 with 95% humidity. Then the cell viability was detected by the Promega CellTiter-Glo® Luminescent Cell Viability Assay (CAT# G7570), which is based on quantitation of the ATP present, an indicator of metabolically active cells. The Luminescence was recorded by the BioTek synergy multi-mode reader (Winooski, VT). The killing percentage was calculated by the formula: (1-Lumi with compounds/Lumi without compounds) ×100. The GraphPad Prism9 for was used for calculation of killing IC50. All experiments were performed in duplicate and the error bars denote ±1 SD.