3e). exhibited potent target-dependent cytotoxicity against polySia-positive tumor cells and and induced quick internalization of polySia into endosomal and lysosomal compartments. In light of these findings, we hypothesized that this antibody-induced endocytosis of polySia-receptors could be efficiently harnessed as part of an antitumor therapeutic strategy. To test this notion, we designed an ADC using a bioorthogonal reaction plan for stably linking the chimeric human ch735 mAb to the microtubule-inhibitory agent maytansinoid DM1, which has previously been developed as the cytotoxic Rabbit polyclonal to PITPNM2 payload in trastuzumab emtansine (T-DM1) for HER2-positive breast malignancy (27). The producing conjugate was found to exert potent target-dependent cytotoxicity against polySia-positive tumor cells = 3). (b) Time course of antibody internalization in polySia-positive cell collection SH-SY5Y treated with ch735 or isotype control. Data reported as the mean percent internalization and error bars are the standard deviation of the mean (= 3). (c) Confocal microscopy images of SH-SY5Y cells incubated for 1 h with ch735 labeled with AF488 and transferrin labeled with AF647. Nuclei were stained by Hoescht (blue). Level bar, 10 m. Fluorescence intensity was measured across the dotted white collection and normalized to the maximum value in each channel. White arrows show regions of colocalization. The inset shows only the ch735 (green) and DNA (blue) channels of the boxed region. (d) Confocal microscopy images of SH-SY5Y cells incubated for 1 h with ch735 labeled with AF488 and anti-LAMP-3 labeled with AF647. Nuclei were stained by Hoescht (blue). Level bar, 10 m. Fluorescence intensity was measured across the dotted white collection and normalized to the maximum value in each channel. White arrows show regions of colocalization. The inset show only the ch735 (green) and DNA (blue) channels of the boxed region. (e) Confocal microscopy images of SH-SY5Y cells incubated for 120 min with ch735. Lysosomes were stained with anti-LAMP-1 and A647-labeled anti-rabbit antibody (reddish), ch735 was stained with AF488-labeled anti-human antibody (green), and nuclei were stained by Hoescht (blue). Level bar, 10 m. Fluorescence intensity was measured across the dotted white collection and normalized to the maximum value in each channel. White arrows show regions of colocalization. The top right inset shows only the ch735 (green) and DNA (blue) channels of the boxed region. Confocal microscopy was used to investigate the compartments where the ch735 mAb accumulated after internalization using markers of early endosomes, recycling endosomes or late endosome/lysosomes. Consistent with circulation cytometry, ch735 in the beginning labeled the plasma membrane of SH-SY5Y cells and after 1 h at 37C was internalized, where it clearly colocalized with early endosomal and recycling endosomal marker transferrin (Fig. 3c) and late endosomal marker LAMP-3 (Fig. 3d). Accumulation of the ch735 mAb was also observed in late endosomal/lysosomal LAMP-1-positive compartments (Fig. 3e). As expected, no detectable binding, internalization or colocalization was observed for the isotype Biotin-X-NHS control (Supplementary Fig. 9a). Much like ch735, the mo735 mAb compartmentalized in early and recycling endosomes as confirmed by colocalization with transferrin and LAMP-3 (Supplementary Fig. 9b and c). Based on these data, we conclude that mAb ch735 binds to tumor cell membranes Biotin-X-NHS in a target-specific manner, thereby inducing a subpopulation of bound antibodies to become rapidly internalized in endosomal/lysosomal compartments. Glycan-directed ADC is usually cytotoxic against tumor cells expressing polySia. Given that ch735 induced internalization of polySia receptors in malignancy cells, we next evaluated whether drug conjugation could Biotin-X-NHS be used to.