Article Figures & Data
Figures
- Figure 1.
Production of recombinant rGel-based immunotoxins targeting FGFR3. A, schematic representation of anti-FGFR3/rGel fusion toxins. VH and VL represent variable region heavy and light chains, respectively, of the FGFR3-specific scFv from clone 3C; (G4S)3, linker consisting of four glycine and one serine residues repeated three times. 218L and SSGSGSA or G4S are different linkers between VL and VH and between scFv and rGel, respectively. B, Coomassie-stained SDS-PAGE under reducing conditions of the purified FGFR3-specific scFv 3C, immunotoxins 3C/rGel and 3CF/rGel, and rGel alone. C, affinity constants of immunotoxins compared with the parental scFv 3C by surface plasmon resonance (BIAcore). The response in resonance units (RU) was recorded as a function of time. Sensorgram overlays show the binding of constructs to immobilized FGFR3 on CM5 chips measured at a constant flow rate of 20 μL/min. The curves are labeled with the samples tested: 3C, 3C/rGel, 3CF/rGel, and rGel alone as a negative control. Inset, affinity constants (Kd in nmol/L) for each construct.
- Figure 2.
In vitro cytotoxicity and antibody-mediated internalization of FGFR3-specific immunotoxins. A, FGFR3-overexpressing RT112 cells were treated with various concentrations of 3C/rGel, 3CF/rGel, or free rGel for 72 h. Cell viability indicates the percentage of cells in drug-treated wells compared with that of control (untreated) wells. Points, mean of triplicate determinations; bars, SD. B, FGFR3 silencing by using a pool of siRNA duplexes specific for FGFR3. RT112 cells were either transfected with nonspecific siRNA [si(nonspecific)] or with FGFR3-specific siRNA [si(FGFR3)] duplexes as described in Materials and Methods. Left, FGFR3 transcript levels were measured by real-time reverse transcription-PCR at 24 h post-transfection. Results are given as percentage of the average of the FGFR3 mRNA level in si(FGFR3)-transfected cells (gray column) relative to that in si(nonspecific)-transfected cells (black column), which corresponded to 100%. Bars, SD. Right, Western blot analysis on lysates of the same aliquots of siRNA-transfected cells confirmed that si(FGFR3) significantly inhibited FGFR3 protein expression. α-Tubulin was detected as a loading control. C, cytotoxicity of 3C-rGel on FGFR3-silenced cells. RT112 cells transfected with si(FGFR3) (♦), si(nonspecific) (▪), or mock (▴) were treated with various concentrations of 3C/rGel (solid lines) or free rGel (dashed lines) at 24 h post-transfection. After 72 h of exposure, cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Cell viability indicates the percentage of cells in drug-treated wells compared with that of control (untreated) wells. Points, mean of three replicates; bars, SD. D, antibody-mediated internalization into RT112 cells was visualized by confocal microscopy. RT112 cells were incubated for 1 h with 50 nmol/L of the corresponding immunotoxin, 3C/rGel or 3CF/rGel, or rGel and saline as controls. After surface-bound proteins were stripped by acid wash, the cells were then processed and stained for rGel using a rabbit polyclonal anti-rGel antibody followed by Cy3-coupled anti-rabbit IgG as detection reagent. Nuclei were counterstained with DAPI. Images were taken with a confocal microscope at ×63 amplification. Bar, 8 μm.
- Figure 3.
Apoptosis induction in FGFR3-expressing bladder carcinoma cell lines by scFv and immunotoxins. A, RT112 cells were treated with 0.2 μmol/L of each construct and, at the indicated time point, were stained with Annexin V-FITC and PI followed by flow cytometry analysis. Numbers in the top of quadrants of each plot represent the percentage of the Annexin V–positive or Annexin V–negative and PI-positive or PI-negative cells. Bottom, percentage of Annexin V–positive cells at 24, 48, and 72 h for each construction tested. B, Western blot showing the level of FGFR3 expression in RT4 cells compared with that in RT112 cells using FGFR3-specific antibodies. α-Tubulin was detected as a loading control. C, RT4 cells were treated with 0.2 μmol/L 3C-rGel or rGel as a control and, at the indicated time point, were stained with Annexin V-FITC and PI followed by flow cytometry analysis.
- Figure 4.
In vivo antitumor activity of the FGFR3-specific scFv and immunotoxins on established RT112 xenografts. Groups of severe combined immunodeficient mice bearing RT112 tumors were treated i.v. with rGel-derived immunotoxins at 50 mg/kg, scFv 3C and free rGel at 25 mg/kg, or saline in alternating days for 2 wk and tested for tumor growth delay of established tumors. A, tumor volumes were measured with calipers about twice a week. Data show two independent experiments each done with four to six mice. Points, mean tumor volume versus time; bars, SD. Dotted line, endpoint size of tumors (1,500 mm3). X axis, arrows, inoculation days. B, percentage of surviving mice are graphed over time.
- Figure 5.
Histopathology analysis of 3C/rGel-treated RT112-derived tumor xenografts. 3C/rGel- and saline-treated tumors were excised at 25 d post-implantation and, 1 d after the final dose, formalin-fixed, paraffin-embedded, and stained for histopathology characterization. A, H&E tumor sections (magnification, ×200) showed more extensive areas of necrosis (arrowhead) in 3C/rGel-treated animals than in the saline control. B, CD34 staining of tumor sections was used to assess blood vessel density (brown, endothelial cells; magnification, ×200). C, Ki-67 staining was used to assess tumor cell proliferation (brown, proliferating cells; magnification, ×400). Columns, mean percentage of proliferating (Ki-67-positive) cells; bars, SD. D, caspase-3-active immunostaining was used to assess the influence of 3C/rGel treatment compared with saline on apoptosis (brown, apoptotic cells; magnification, ×400). Columns, mean percentage of apoptotic (caspase-3-active-positive) cells; bars, SD. *, P < 0.0001 (Student's t test).
- Figure 6.
Immunolocalization of FGFR3 in 3C/rGel- and saline-treated RT112-derived tumors. 3C/rGel- and saline-treated tumors were excised at 25 d post-implantation and, 1 d after the final dose, formalin fixed, paraffin-embedded, and stained with anti-FGFR3 antibody. A, tumor sections of RT112-derived xenografts treated with either 3C/rGel (top) or vehicle (bottom) were used to evaluate the presence and location of FGFR3 on tumor cells by immunohistochemistry (brown, FGFR3-positive cells; left, magnification, ×100; right, magnification, ×400). B, immunofluorescence with anti-FGFR3 antibody (green) of tumor sections from 3C/rGel-treated showing nuclear staining (top) and nontreated with cytoplasm staining (bottom) xenografts (magnification, ×200). Sections were counterstained with DAPI (blue) to visualize the nuclei. Column 3, merged images of the labeled cells. Insets, a close-up with intense nuclear staining.
Volume 7, Issue 4
