Article Figures & Data
Figures
- Figure 1
Preparation and characterization of scFv constructs. (A) Scheme of the constructs for anti-FAP scFv, anti-FAP scFv-IL-872 and anti-FAP scFv-IL-83-72 as they were inserted into the mammalian expression vector pEAK8. The anti-FAP scFv antibody vector consisted of the promoter-leader cassette coding for an 18-aa secretory leader sequence, the variable heavy chain region followed by a 10-aa linker with the light chain variable region and a DNA sequence encoding a c-myc and His6 tag at the C-terminus for detection and affinity purification. IL-8 variants are N-terminally linked to anti-FAP scFv by (Ser4Gly)3. (B) Anti-FAP scFv-IL-8 (lanes 1 to 3) and anti-FAP scFv-IL-83-72 (lanes 4 to 6) were purified by immobilized metal chelate chromatography. Culture supernatant (lanes 1 and 4), flow through (lanes 2 and 5) and purified constructs (lanes 3 and 6) were analyzed by SDS-PAGE under reducing conditions. As expected, bands corresponding to scFv IL-8 variants were detected at 39 kDa (lane 3 for anti-FAP scFv-IL-872 and lane 6 for anti-FAP scFv-IL-83-72). (C) Western blot analysis of anti-FAP scFv (lanes 1 and 4), anti-FAP scFv-IL-872 (lanes 2 and 5) and anti-FAP scFv IL-83-72 (lanes 3 and 6) stained with anti-c-myc (lanes 1 to 3) and anti-hu IL-8 antibody (lanes 4 to 6).
- Figure 2
Binding properties of scFv constructs. (A) Flow cytometry analysis of constructs [anti-FAP scFv (thick line), anti-FAP scFv-IL-83-72 (dotted line) and anti-FAP scFv-IL-872 (thin line)] was performed on FAP-transfected HT1080 cells. An anti-CD30 scFv-IL-8 fusion protein served as negative control. Binding was detected by monoclonal anti-c-myc antibody. (B) Recognition of targeted antigen by scFv constructs was performed by incubation with rabbit anti-human IL-8 and visualized by PE-conjugated goat anti-rabbit serum. Uncoupled parental anti-CD30 scFv-IL-8 was used as negative control. (C) Binding of scFv variants to immobilized anti-FAP-anti-ID was visualized by rabbit anti-IL-8 serum using ELISA. Uncoupled anti-FAP scFv and anti-CD30 scFv-IL-8 were used as controls. Measurements were carried out in triplicate samples; standard deviations are indicated by the bars.
- Figure 3
Chemotactic potential of anti-FAP scFv-IL-8 variants in vitro. We used a fluorescence-based end-point assay to measure PMN migration in vitro. Chemo-attractants (rhu IL-8, anti-FAP scFv, anti-FAP scFv-IL-83-72 and anti-FAP scFv-IL-872) and controls were diluted in PBS-HSA 0.1%. PBS-HSA 0.1% alone was used to determine random migration. In order to define the total fluorescence of PMNs, calcein-AM labeled PMNs were placed directly at defined concentrations (80000, 8000, 800 and 80 cells) in three separate wells. 80000 PMNs were placed directly onto the top of the filter and the chamber was incubated for 60 minutes. The total number of PMNs that had migrated to the lower chamber was measured with a fluorescence plate reader. Measurements were carried out in triplicate samples. The standard deviation of three assays is shown.
- Figure 4
Kinetics of FAP expression of transfected HT1080-FAP+ fibrosarcoma cells in vitro and in vivo. (A) FAP transfected HT1080 cells were cultured without G418 selective pressure and the levels of antigen density were measured after 3 weeks by FACScan analysis. (B to E) Immunohistochemical staining of HT1080-FAP+ xenograft fibrosarcoma cells showing positive staining over a period of four weeks. 5 x 106 cells were injected s.c. in BALB/c nu/nu mice. Solid tumors were harvested after 14 (B, C) and 28 days (D, E), respectively. Tissue sections were stained with anti-FAP IgG (B, D) or with anti-G250 IgG (C, E) as a control. Bars correspond to 10 µm.
- Figure 5
Targeted antigen-dependent recruitment of PMNs to FAP-expressing tumors. Fibrosarcomas were established in BALB/c nu/nu mice by s.c. injection of 5 x 106 FAP-transfected and mock-transfected HT1080 cells, respectively. FAP-positive (left column) and -negative (right column) tumors were simultaneously established at the opposite flanks of the animals. Tumors were harvested 24 hours after i.v. injection of a total dose of 300 µg scFv-IL872 (equivalent to 100 µg rhu IL-8). (A, B) FAP expression of xenografts was confirmed by anti-FAP mAb staining. PECAM-1-positive tumor vessels were visualized to define comparable areas of FAP-positive (C) or -negative (D) fibrosarcomas to exclude unspecific PMN accumulation. Staining with anti-CD11b mAb demonstrated the targeted antigen-specific delivery of scFv-IL872 by chemokine-triggered enrichment of PMNs (E, F). Bars correspond to 100 µm.
- Figure 6
Effects of sequential immunotherapy on the growth pattern of tumor xenografts. (A) FAP positive tumors were established in BALB/c nu/nu mice by s.c. injection 5 x 106 HT1080 FAP+ cells and mice randomly assigned to different treatment groups at day nine. For sequential immunotherapy, effector constructs were injected i.v. and followed eight hours later by i.v. injection of the second effector construct as indicated (100 µl per injection). This treatment regimen was repeated every three days as highlighted by the arrows (five times in total). Starting on day 21, the growth delay was statistically significant for the anti-FAP TNF & anti-FAP scFv-IL-872 group (*, P < 0.001) when compared to all other treatment groups. Comparison of tumor sizes between the anti-FAP TNF and the anti-FAP TNF & rhu IL-8 group and the remaining groups was also significant at this time point (**, P < 0.001), but tumor sizes did not differ significantly between the anti-FAP TNF and the anti-FAP TNF & rhu IL-8 group. Single agent treatment with anti-FAP scFv-IL-872 also resulted in significant (***, P < 0.001) anti-tumor effects when compared to the rhu IL-8-containing regimens that did not differ from the PBS group (data not shown). Treatment results stayed significant over the observation period. Animals were taken off study when tumor volume exceeded 1 cm3. (B) Delay of tumor growth of mice receiving anti-FAP TNF & anti-FAP scFv IL-8 was linked to antigen expression. Simultaneous growth of two phenotypically different xenografts [FAP-positive (closed symbols) and FAP-negative (open symbols)] was established in nude mice and animals assigned to their respective treatment group when tumor diameter reached 5 mm. Again, animals were treated with anti-FAP TNF & anti-FAP scFv-IL-872 following the sequential application schedule indicated by the arrows. Mice had to be taken off study when the volume of FAP-negative xenografts exceeded 1 cm3. At this time point, differences in tumor size between FAP-positive and -negative xenografts were statistically significant (*, P < 0.001).
Volume 9, Issue 1
