Figure 1.
Selection and purification of L1 scFvs from the Tomlinson I library.
(A) Binding of monoclonal phages to substrate-coated L1/ecd was determined by ELISA. Phages (1×109 cfu) from the third selection round were incubated with L1/ecd. Substrate-coated BSA was used as negative control. Bound phages were detected with anti-M13 antibody conjugated to HRP. (B) Expression and purification of scFvs. Lane 1, bacterial supernatant containing scFvs; lane 2, column flow-through; lane 3, affinity chromatography purified scFv I27 (indicated by arrow) following column elution with 100 nM imidazole. Molecular weight markers are indicated at the left margin. (C) Amino acids sequences of complementarity determination regions 2 and 3 on heavy and light chains of L1 scFvs. Different amino acids are underlined.
Figure 2.
Binding of purified scFvs to L1.
Binding of purified scFvs to L1/ecd, Ig1–4, Fn1–3 was determined by ELISA. (A) ScFv I4, (B) scFv I6, (C) scFv I13, (D) scFv I27. ScFvs were incubated with substrate-coated L1/ecd, Ig1–4, Fn1–3, human Fc fragment, or bovine serum albumin (BSA) at the indicated concentrations. Bound scFvs were detected with biotinylated antibodies against c-myc followed by incubation with ExtrAvidin-HRP. Data represent mean values ± SEM from 3 independent experiments.
Figure 3.
Immunostaining of live SK-N-SH cells with ScFvs.
Substrate-attached SK-N-SH cells were incubated with scFvs I4 (A), I6 (B), I13 (C), or I27 (D), or without scFv (E). Bound scFvs were visualized by rabbit antibody against the His-tag followed by incubation with Alexa 488 nm (green)-conjugated goat anti-rabbit IgG and double labeling with goat anti-human extracellular domain of L1 as positive control, followed by Alexa 594 nm (red)-conjugated donkey anti-goat IgG. Bar in (E) indicates 5 µm for all panels. (F) Binding of purified scFvs I4, I6, I13, I27 to an SK-N-SH cell lysate was tested by subjecting 50 µg protein to SDS-PAGE in 8% gels under reducing conditions. Western blot analysis was carried out with scFvs I4, I6, I13, I27, and goat anti-human L1 as primary antibodies. Primary antibodies were detected with secondary antibody against His or rabbit anti-goat, respectively. Molecular weight markers are indicated at the left margins in kilodaltons (kDa).
Figure 4.
ScFvs against Ig1–4 inhibit, while scFvs against Fn1–3 increase proliferation of SK-N-SH cells.
Representative images are merged with bright field, DAPI (blue) nuclear staining, and Ki67 immunostaining (red). (A) Control without treatment, (B) negative control with non-immune human IgG (10 µM), (C) scFv I4, (D) scFv I6, (E) scFv I13, (F) scFv I27, (G) positive control with L1/ecd (all at 16.5 µM). Scale bar in (E) indicates 20 µm (for all panels). (H) Mean values ± SEM of the ratio of Ki67-immunopositive cells to DAPI-positive cells are shown from three independent experiments. Asterisks denote significant differences from control. *** p<0.001, ** p<0.01, * p<0.05, Student's t-test.
Figure 5.
ScFvs against Ig1–4 inhibit, while scFvs against Fn1–3 stimulate transmigration of SK-N-SH cells.
Equal numbers of cells (105) were seeded into the upper compartment of a transwell chamber and allowed to migrate through a BSA-coated membrane for 36 hours. (A) Cells that had migrated into the lower chamber containing L1/ecd (12 µM), non-immune human IgG (10 µM), and scFvs I4 (16.5 µM), I6 (16.5 µM), I13 (16.5 µM), or I27 (16.5 µM) are shown. Omission of scFvs served as negative control. Migrated cells were fixed on the membrane and lower chamber, stained with crystal violet and counted. (B) Non-transmigrated cells in the upper chamber were also counted. Data represent mean values ± SEM of numbers of transmigrated cells from three independent experiments. Asterisks denote significant differences from control. ** p<0.01, * p<0.05 versus the number of cells in the control, Student's t-test.
Figure 6.
ScFvs against Ig1–4 inhibit, while scFvs against Fn1–3 stimulate neurite outgrowth from SK-N-SH cells.
Human SK-N-SH cells were seeded into poly-D-lysine (PDL)-pretreated culture plates coated with human non-immune human IgG (10 µM), L1/ecd (3 µM), scFvs I4, I6, I13 and I27 as substrates at the indicated concentrations and cultured at 37°C. Cultures were fixed after 5 days and stained with toluidine blue. The lengths of neurites were measured. Data represent mean values of neurite lengths per cell ± SEM as compared with wells coated with PDL only from three independent experiments. Asterisks denote significant differences from control. *** p<0.001, ** p<0.01, * p<0.05, Student's t-test.
Figure 7.
ScFvs against Fn1–3 protect cells from H2O2–induced death and increase the ratio of Bcl-2/Bax.
SK-N-SH cells were seeded into poly-D-lysine (PDL)-pretreated culture plates coated with L1/ecd (as positive control), non-immune human IgG (as negative control), and scFvs I4, I6, I13, or I27 and maintained for 5 days in serum-free medium. In addition, SK-N-SH cells grown for 12 hours were pre-incubated with 250 µM H2O2 for 2 hours in serum-free medium. After removal of H2O2, scFvs I13 and I27 were added at the indicated concentrations and cells were cultured for additional 24 hours. (A) ScFvs treatment for 5 days. Cell survival was measured by the MST-8 assay. (B) L1/ecd, non-immune human IgG, scFvs I4, I6, I13 or I27 (all at 16.5 µM) treatment for 24 hours for Western blot assay of Bcl-2 and Bax protein expression. (C) ScFv treatment for 12 hours. (D) ScFv treatment for 24 hours. (C, D) Cell viability was measured by the MST-8 assay. Data represent mean values ± SEM from four independent experiments. Asterisks denote significant differences from control. *** p<0.001, ** p<0.01, * p<0.05 versus H2O2 treatment alone, one-way ANOVA, Dunnet's post-hoc test. (E) ScFvs I4, I6, I13 and I27 (all at 16.5 µM), were added after removal of H2O2 for 24 hours. Cells were stained by Hoechst 33342 (blue) and propidium iodide (PI, red), merge (pink). Bar indicates 40 µm for all panels. (F, G) Quantification of PI and Hoechst positive cells, and calculation of the number of PI and Hoechst positive cells among the total cell number. (B, H) Analysis of protein levels of Bcl-2/Bax after treatment of cells with scFvs. GAPDH was used as loading control. (B, E, F, H) Data represent mean values ± SEM from three independent experiments. *** p<0.001, ** p<0.01, * p<0.05, Student's t-test.
Figure 8.
ScFvs against Ig1–4 reduce, while scFvs against Fn1–3 enhance levels of phospho-src and downstream phospho-Erk1/2.
Cultured SK-N-SH cells were incubated with L1/ecd (as positive control), non-immune human IgG (as negative control), and scFvs I4, I6, I13, or I27 for 30 min in serum-free medium. (A) Levels of src and phospho-src of cells treated with L1/ecd (at 16.5 µM, right) or untreated (control, left), with lanes 1, 2 and 3 showing representative blots from three independent experiments with L1/ecd treated or untreated cells. (B) Src and phospho-src levels of cells treated with non-immune human IgG, L1/ecd or I27 (all at 16.5 µM) or left untreated (control). (C) Src and phospho-srclevels of cells treated with the indicated concentrations of scFv I4 or I6. (D) Src and phospho-src levels of cells treated with the indicated concentrations of scFv I13 or I27. (E–F) Comparison of levels of src and phospho-src (E) and Erk1/2 and phospho-Erk1/2 (F) in untreated cells (control) or cells treated with IgG, scFv I4, I6, I13 or I27 (all at 16.5 µM). Representative images from three independent experiments are shown in A–E. For the quantification shown in C–F, values of phospho-src (C–E) and phospho-Erk1/2 (F) were normalized to GAPDH as loading control. Mean values ± SEM from three independent experiments are shown. Asterisks denote significant differences from control. ** p<0.01, * p<0.05 by Student's t-test.
Table 1.
Binding properties and effects of the different scFvs.
Figure 9.
Signal transduction events following exposure of neuroblastoma cells to scFvs reacting with L1.
ScFvs binding to the Ig1–4 domain of L1 in neuroblastoma cells inhibit phosphorylation of src and Erk most likely through disruption of homophilic or heterophilic L1 interactions. ScFvs binding to the Fn1–3 domain (such as the Fn3 domain of L1) increase phosphorylation of src and Erk by triggering of L1-L1 or L1-FGFR/L1-integrin signaling.