Additional file 2 of Subcellular trafficking and transcytosis efficacy of different receptor types for therapeutic antibody delivery at the blood‒brain barrier
Creators: name: Holst, Mikkel Roland
name: de Wit, Nienke Marije
name: Ozgür, Burak
name: Brachner, Andreas
name: Hyldig, Kathrine
name: Appelt-Menzel, Antje
name: Sleven, Hannah
name: Cader, Zameel
name: de Vries, Helga Eveline
name: Neuhaus, Winfried
name: Jensen, Allan
name: Brodin, Birger
name: Nielsen, Morten Schallburg
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Additional file 2: Figure S1. Verification of cargo antibody specificities. (A-D) Western blotting analysis of antibody specificity indicated by single bands in human and mouse lysates (6 μg loaded per lane). (E) The diagram illustrates how parent bivalent K409R or F405L mutated antibodies were resembled into bispecific monovalent antibodies for this study using controlled fab-arm exchange. (F-I) Immunofluorescence total staining of hiPSC-BECs. Humanized bispecific antibody construct (Genentech 15G11 origin) targeting HIV G120 (B12) and TfR shows single band detection in human cell lysates (A) and binding to SBAD-02-01 expressed TfR (F). Both mouse anti-sortilin (B and G) and mouse anti-podocalyxin (C and H) show specificity to their human antigens with cross-species detection towards mouse variants in mouse capillary lysates (B and C). Mouse anti-CD133 shows specific detection of human CD133 (D and I) with no detection of mouse variants in bEnd.3 or mouse capillary lysates (D). Scale bars are 10 μm. Figure S2. Effect of medium change on barrier tightness for transport experiments. (A-B) Total receptor stains showing receptor localization after changing medium into transport medium. Cells are stained for ZO1 in white and the indicated receptors in green. (C-D) TEER measurements before medium change (Start), three hours after medium change (three-hour time-point for addition of cargo antibody spike solution) and 24 hours (24 hours after cargo antibody spike time point). TEER values were measured using an EVOM Epithelial Volt/Ohm meter (World Precision Instruments, Friedberg, Germany) with chopstick electrodes. The TEER measurement of a cell-free collagen IV/fibronectin-coated filter was used as blank, which was subtracted from TEER values measured on filter-seeded cells. Bar plots show mean values ±SD based on three independent experiments. (F) Sodium fluorescein (NaF) was used to measure paracellular permeability through the hiPSC-BEC layer. The box plot show mean value ±SD of three independent experiments. (F) Representative micrographs of four selected endothelial markers stained with antibodies described before (Hudecz et al., 2023). See also supplementary table 1 for RNA expression analysis. Scale bars are 10 μm. Figure S3. Positioning of cargo antibodies in cell layers. (A-B) Micrographs show side views (x-z) of maximum projected z-stacks. Cells are stained for ZO1 in white and the indicated receptors in green. Sorting receptors TfR and sortilin are both expressed in hiPSC-BECs and Caco-2 cells and are localized at intracellular surfaces. The polarized receptors podocalyxin and CD133 are both expressed in the apical surface facing the luminal side of the Transwell chamber. The dashed blue lines in (A-B) indicates the filter surface (abluminal cell surface). Scale bars are 5 μm. (C-D) Receptor expression (total stain) and cargo antibody signals were analysed using immunofluorescent spot analysis for the indicated receptors in hiPSC-BECs (C) and similarly in for Caco2 cells in (D). All bar plots show Mean ±SD based on three independent experiments. Figure S4. Representative control immunostainings for markers and cargo receptors. (A) Representative maximum projected z-stack micrographs from stains against selected markers of the study. (B-C) Maximum projected z-stack of micrographs from total receptor stains showing receptor localization after changing medium into transport medium. The x-y areas marked by the white stippled line are shown as x-z side views. Scale bars are 10 μm. Figure S5. Sorting and transcytosis of cargo antibodies through hiPSC-BECs without astrocyte coculture. (A) Colocalization analysis of internalized cargo antibodies with selected markers in relation to the steady-state receptor (total receptor in black bars). (B) ELISA measurement of antibody in the Transwell bottom acceptor chamber in relation to the added antibody in the top donor chamber given as transcytosed IgG percentage through the hiPSC-BEC layer (without prior astrocyte NCC). The red stippled line indicates the lower level of detection (approx. 50 pM) for the ELISA setup. Statistical significance was determined using two-way ANOVA with Tukey´s multiple comparisons test. All bar plots show Mean ±SD based on three or four independent experiments.