Figure 1.
Selection and validation of VHHs against hpIgR.
(A) Outputs of phage display selection on coated recombinant hpIgR ectodomain with either trypsin or IgA elution for llama's 97 and 98. (B) Pull down of VHHs with incubated cell lysate of MDCK control (right) or MDCK-hpIgR (left) cells with the anti-his matrix Talon. Western blot with anti-hSC and anti-myc tag (VHH) (n = 3). (C) A critical amount of IR800 labelled dIgA with indicated amount of VHHs was incubated on MDCK-hpIgR cells at 4°C for 1.5 h. The normalized amount of IR800-dIgA is calculated and set out in the graph against the VHH concentration (n = 4) (s.e.m.'s are shown for all points). (D+E) Pull down of VHHs incubated in MDCK-hpIgR cultured medium containing hSC (D) or with 4 µg/ml sIgA (E). Western blot with anti-hSC, anti-hIgA heavy chain and anti-myc (n = 4). (F) VHHs were titrated and put on cells for 1.5 h at 4°C. VHH presence was quantified by using anti-myc tag and D-α-M-IR800 antibodies. Quantification as described in experimental procedures. KD determinations were done with a ‘one-site specific binding’ algorithm in Graphpad. (n = 6).
Figure 2.
Selected VHHs internalize into and transcytose across polarized epithelial cells expressing hpIgR.
(A) 1 µM of 4b7 VHH was applied either basolaterally (for 20 min) or apically (for 60 min) on polarized MDCK-hpIgR cells and visualized after fixation with an anti-myc tag and G-α-M-Alexa488 antibody and DAPI staining both in xy and xz plane (n = 4). 1 µM of 4b7 VHH was applied to confluent MDCK control cells and treated as above (n = 4). (B) Internalization assay of selected VHHs. 1 µM VHH was incubated on cells at 37°C for 1, 3 or 7 min. Externally bound VHH was removed with 25 µg/ml trypsin for 45 min on ice. Internalized VHH was immunolabelled with an anti-myc tag and D-α-M-IR800 antibodies. Quantification of fluorescence is described in experimental procedures. Fluorescence intensities of the internalized amount is divided over the amount of cells (To-pro3 nuclear stain) and plotted against time. The signal of the irrelevant anti-hTfR VHH was deducted from the specific VHH signal and therefore represents the x-axis (n = 12) (s.e.m.'s are shown for all points). (C) Transcytosis assay using VHH displaying phages. Phages were applied basolaterally and incubated for 5 h. Amount of phages in apical medium is subsequently quantified as described in experimental procedures and plotted per construct (n = 4) (s.e.m.'s are shown for all points). Significance measurements were done by two-way ANOVA.
Figure 3.
Myc-tagged VHHs were incubated with increasing amounts of flag-tagged VHHs in an hpIgR coated well for 2 h at room temperature. Myc tag presence was subsequently quantified and competition therewith assessed. Competition is depicted in a chequer board diagram were black indicates competing VHHs (n = 2). See Figure S2 for raw data.
Figure 4.
Bivalency enhances internalization.
(A–D) Internalization assay of selected VHHs. 1 µM VHH was incubated on cells at 37°C for 1, 3 or 7 min. Externally bound VHH was removed with 25 µg/ml trypsin for 45 min on ice. Internalized VHH was immunolabelled with an anti-myc tag and D-α-M-IR800 antibodies. Quantification of fluorescence is described in experimental procedures. Fluorescence intensities of the internalized amount is divided over the amount of cells (To-pro3 nuclear stain) and plotted against time. The signal of the irrelevant anti-hTfR VHH was deducted from the specific VHH signal and therefore represents the x-axis (n = 12) (s.e.m.'s are shown for all points).
Figure 5.
Transcytotic capacity of optimized constructs.
(A+B) 1 nM 125I radio-labelled VHH or dIgA was applied in the basolateral chamber of polarized MDCK-hpIgR cells (A) or Caco-2 cells (B) grown on transwell inserts. Apical medium was resorbed after each 2 h interval to a maximum of 6 h and quantified separately. Amount of pmol that is transcytosed is plotted against time. Leakage of a non-specific VHH control was deducted from the specific signal (n = 4) (s.e.m.'s are shown for all points). Significance measurements were done by two-way ANOVA.