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Video 1. Time-lapse DIC Imaging. Time-lapse sequential DIC images (video) showed swelling and then shrinkage of the cell after NIR light irradiation in cells treated with tra-IR700 (50 min observation). Flashing light is NIR light irradiation (2 J/cm2).(10sec interval, total 50min)

Supplementary Figures 1-5. Video Captions (Video 1 and 2) Supplementary Figure 1. Targeted necrotic cell death observed after NIR-PIT in vitro. Supplementary Figure 2. Necrotic cell death evaluated on serial bioluminescence imaging after NIR-PIT Supplementary Figure 3.In vivo tumor inhibition by NIR-PIT in flank model Supplementary Figure 4. In vivo fluorescence imaging of tumor bearing mice before and after NIRPIT Supplementary Figure 5. Mesothelin targeted necrotic cell death observed after NIR-PIT in vitro

Video 2. Time-lapse PI Imaging. Time-lapse sequential PI images (video) showed rapid membrane damage detected by PI staining after NIR light irradiation in cells treated with tra-IR700 (50 min observation). Flashing light is NIR light irradiation (2 J/cm2).(10sec interval, total 50min)

Table S1. Biodistribution of radioactivity in mice after injection of 67Ga-NOTA-Fab, 67Ga-NOTA-MI-Fab and 67Ga-NOTA-MVK-Fab. Table S2. Biodistribution of radioactivity in nude mice bearing SY cells after 3 h injection of 67Ga-NOTA-Fab, 67Ga-NOTA-MI-Fab and 67Ga-NOTA-MVK-Fab. Table S3. Stability of 67Ga-labeled Fab fragments in murine serum. Scheme S1. Synthetic procedure for NOTA-MVK-Mal. Scheme S2. Synthetic procedure for NOTA-MVK-Bzo.

Purpose: This study was undertaken to evaluate the renal radioactivity levels of a newly designed 67Ga-labeled antibody fragment with a linkage cleaved by enzymes present on the brush border membrane (BBM) lining the lumen of the renal tubule.Experimental Design: 67Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) was conjugated with an antibody Fab fragment through a Met-Val-Lys linkage (67Ga-NOTA-MVK-Fab) considering that a Met-Val sequence is a substrate of enzymes on the renal BBM and 67Ga-NOTA-Met is excreted from the kidney into the urine. The enzymatic recognition of the linkage was evaluated with a low-molecular-weight 67Ga-NOTA-Met-Val-Lys derivative. Biodistribution of radioactivity after injection of 67Ga-NOTA-MVK-Fab into mice was compared with 67Ga-NOTA-conjugated Fab fragments through a Met-Ile linkage that liberates 67Ga-NOTA-Met (67Ga-NOTA-MI-Fab) or a conventional thiourea linkage (67Ga-NOTA-Fab).Results: The MVK linkage remained stable in plasma and was recognized by enzymes on renal BBM to liberate 67Ga-NOTA-Met. When injected into mice, all three 67Ga-labeled Fab exhibited similar blood clearance rates and tumor accumulation. Significant differences were observed in the kidney where 67Ga-NOTA-MVK-Fab registered the lowest renal radioactivity levels from early postinjection time (P < 0.05), followed by 67Ga-NOTA-MI-Fab, which was well reflected in the SPECT/CT images.Conclusions: These findings indicated that our proposal of liberating a radiolabeled compound to urinary excretion from antibody fragments at the renal BBM to reduce the renal radioactivity levels was applicable to 67/68Ga-labeled antibody fragments. Because antibody fragments and constructs share similar metabolic fates in the kidney, the present labeling procedure would also apply to a variety of antibody fragments and constructs of interest. Clin Cancer Res; 24(14); 3309–16. ©2018 AACR.

Purpose: This study was undertaken to evaluate the renal radioactivity levels of a newly designed 67Ga-labeled antibody fragment with a linkage cleaved by enzymes present on the brush border membrane (BBM) lining the lumen of the renal tubule. Experimental Design: 67Ga-labeled S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (SCN-Bn-NOTA) was conjugated with an antibody Fab fragment through a Met-Val-Lys linkage (67Ga-NOTA-MVK-Fab) considering that a Met-Val sequence is a substrate of enzymes on the renal BBM and 67Ga-NOTA-Met is excreted from the kidney into the urine. The enzymatic recognition of the linkage was evaluated with a low-molecular-weight 67Ga-NOTA-Met-Val-Lys derivative. Biodistribution of radioactivity after injection of 67Ga-NOTA-MVK-Fab into mice was compared with 67Ga-NOTA-conjugated Fab fragments through a Met-Ile linkage that liberates 67Ga-NOTA-Met (67Ga-NOTA-MI-Fab) or a conventional thiourea linkage (67Ga-NOTA-Fab). Results: The MVK linkage remained stable in plasma and was recognized by enzymes on renal BBM to liberate 67Ga-NOTA-Met. When injected into mice, all three 67Ga-labeled Fab exhibited similar blood clearance rates and tumor accumulation. Significant differences were observed in the kidney where 67Ga-NOTA-MVK-Fab registered the lowest renal radioactivity levels from early postinjection time (P < 0.05), followed by 67Ga-NOTA-MI-Fab, which was well reflected in the SPECT/CT images. Conclusions: These findings indicated that our proposal of liberating a radiolabeled compound to urinary excretion from antibody fragments at the renal BBM to reduce the renal radioactivity levels was applicable to 67/68Ga-labeled antibody fragments. Because antibody fragments and constructs share similar metabolic fates in the kidney, the present labeling procedure would also apply to a variety of antibody fragments and constructs of interest. Clin Cancer Res; 24(14); 3309–16. ©2018 AACR.