Journal: Molecular cancer therapeutics

Article Title: Manipulation of cell-type selective antibody internalization by a guide-effector bispecific design

doi: 10.1158/1535-7163.MCT-18-1313

Figure Lengend Snippet: A) ALCAM cell surface level post antibody treatment. Pancreatic cancer cell lines (L3.6pl, Capan-1, Panc-1) were incubated with 3F1, 3F1/RYR, C10/RYR, or a mixture of 3F1 and C10/RYR. Following wash post treatment, cell surface ALCAM level was determined using an Alexa® 647-labeled IgG that bind to a different epitope on ALCAM than 3F1. MFI values were normalized against cells without antibody treatment. **P<0.01, and ***P<0.001. Duplicates. B) Confocal microscopy study of cell-type selective internalization mediated by the bispecific. L3.6pl (E/A ratio > 0.2) and Panc-1 (E/A ratio < 0.2) cells were incubated with 3F1, 3F1/RYR, or C10/RYR, and internalizing antibodies were stained with FITC-labeled anti-human IgG. Scale bar: 20 μm. C) Co-localization of antibodies and macropinocytotic vesicles. L3.6pl cells were incubated with 3F1, 3F1/RYR, or C10/RYR at 100 nM and ND70-TR (TR-Dextran, red) for 2 hours. Antibodies were detected by FITC-labeled anti-human IgG (green). Nuclei were labeled with Hoechst 33342 (blue). Scale bar: 10 μm. D) Lysosomal trafficking post internalization. L3.6pl cells were incubated with indicated antibodies (100 nM) for 2 hours. Internalized antibodies (green) and nuclei (blue) were stained as described in C), and lysosomes were detected using rabbit anti-LAMP1 primary IgG, followed by Alexa® 647-labeled anti-rabbit IgG (red). Scale bar: 10 μm. E) Retarded EphA2 internalization on Panc-1 cell when targeted by the bispecific. **P<0.01, and ***P<0.001. Duplicates. F) A time course of EphA2 removal from Panc-1 cell surface at 0.5, 1, and 4 hours post antibody treatment.

Article Snippet: Lysosomes were detected by rabbit anti-lysosomal-associated membrane protein 1 (LAMP1) antibody (Cell Signaling) followed by incubation with Alexa Fluor® 647-labeled goat anti-rabbit IgG (Jackson ImmunoResearch).

Techniques: Incubation, Labeling, Confocal Microscopy, Staining