Figure 2. Western blot analysis of the anti-CD30 antibody-cytokine fusion proteins.

<p>(A) Supernatants containing HRS3-scFv-IL12 and HRS3-scFv-Fc-IL12 or HRS3-scFv-IL2 and HRS3-scFv-Fc-IL2 fusion proteins, respectively, were separated by SDS-PAGE under reducing (R) and non-reducing (NR) conditions. (B) Supernatants of 293T producer cell lines containing HRS3-scFv-Fc-IL2 and dual cytokine HRS3-scFv-IL12-Fc-IL2 fusion proteins, respectively, were separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). Separated proteins were transferred to PVDF membranes and detected by staining the blots with anti-IL12 and anti-IL2 antibodies, respectively.</p

Figure 1. Schematic diagram of the anti-CD30 antibody-cytokine fusion proteins used in the study.

<p>IL12 represents the p35–p40 single-chain IL12. hi: IgG1 hinge region; IgG1-Fc: IgG1 CH2CH3.</p

Figure 4. Biodistribution of HRS3-scFv-IL12 and HRS3-scFv-Fc-IL12 fusion proteins.

<p>HRS3-scFv-IL12 and HRS3-scFv-Fc-IL12 fusion proteins were purified and labeled with ¹³¹I as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044482#s4" target="_blank">Materials and Methods</a>. SCID mice were s.c. transplanted with CD30⁺ L540cy Hodgkin's lymphoma cells (2×10⁷/animal) and animals with established tumors (>0.5 cm³) were intravenously injected with ¹³¹I -labeled fusion proteins. Animals were sacrificed after 24 h, 48 h and 72 h (HRS3-scFv-IL12: 4 mice/group; HRS3-scFv-Fc-IL12: 3 mice/group), respectively, organs were recovered and tissue retention of radioactivity (%) was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044482#s4" target="_blank">Materials and Methods</a>.</p

Biodistribution of HRS3-scFv-Fc-IL12 fusion protein.

1<p>Values represent the mean percentage of injected dose per g tissue (n = 3 mice).</p><p>doi:10.1371/journal.pone.0044482.t002</p

Figure 8. HRS3-scFv-IL12-Fc-IL2 fusion protein improves lysis of CD30⁺ tumor cells and represses tumor growth in immune-competent mice.

<p>(A) CD30⁺ and CD30⁻ MC38 tumor cells were pre-incubated for 1 h on ice with equimolar amounts of the indicated fusion proteins. Cells were washed extensively and co-cultured (5×10⁴ cells/well) for 48 hrs in round-bottom micro-titer plates together with freshly isolated, non-activated NK cells (each 5×10⁴ cells/well). Co-incubation with NK cells without fusion protein (medium) served as control. Cytotoxicity against CD30⁺ tumor cells was determined by a XTT-based assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044482#s4" target="_blank">Materials and Methods</a>. Data represent the mean of triplicates ± standard error of mean (SEM). Statistical analyses were performed by Student's T test. A representative experiment out of three is shown. (B) Balb/c mice were transplanted with 9G10 hybridoma cells expressing the HRS3-specific anti-idiotypic mAb on the cell surface (n = 19) or for control with C10 hybridoma cells expressing an IgG1 with irrelevant specificity (n = 10) (2.5×10⁶ cells/mouse). At day 7, when the tumor was fairly established, mice received i. v. injections of the purified HRS3-scFv-IL12-Fc-IL2 fusion protein (50 µg/animal) or the same volume PBS for control (5–10 animals/group). Injections were repeated twice every second day. Tumor volume was recorded every 2–3 days. Mean values are shown; p values were determined by Student's T test.</p

Biodistribution of HRS3-scFv-IL12 fusion protein.

1<p>Values represent the mean percentage of injected dose per g tissue (n = 4 mice).</p><p>doi:10.1371/journal.pone.0044482.t001</p

Figure 3. Antigen-specific binding of antibody targeted IL-2 and IL12 fusion proteins.

<p>(A) Equimolar amounts of the scFv-Fc fusion proteins were incubated in serial dilutions in micro-titer plates coated with the anti-idiotypic mAb 9G10 which binds to the HRS3 scFv antibody targeting domain of the fusion proteins. For control, same amounts of human IgG1 were added. Bound fusion proteins were detected by an anti-IgG1 antibody directed towards the Fc spacer domain. A representative experiment out of three is shown. (B) CD30⁺ L540cy Hodgkin's lymphoma cells were incubated with serial dilutions of the anti-CD30-Fc fusion proteins or of IgG1 for control. Bound proteins were detected by a PE-conjugated F(ab)₂ goat anti-human IgG1 antibody. Cells were analyzed by flow cytometry and the mean fluorescence intensity (MFI) was determined. A representative experiment out of three is shown. (C) Equimolar amounts of purified HRS3-scFv-IL12 and HRS3-scFv-Fc-IL12 fusion proteins, respectively, or (D) of HRS3-scFv-IL2 and HRS3-scFv-Fc-IL2 fusion proteins, respectively, were incubated in CD30 coated microtiter plates in the presence of increasing amounts of soluble CD30. Bound proteins were detected by biotinylated anti-IL12 (C) or anti-IL2 (D) antibodies and streptavidin-conjugated POD. Binding inhibition was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044482#s4" target="_blank">Materials and Methods</a>. An experiment out of two with similar results is shown.</p

Figure 7. The HRS3-scFv-IL12-Fc-IL2 dual cytokine fusion protein bound to CD30⁺ cells activates resting NK cells to IFN-γ secretion.

<p>CD30⁺ and CD30⁻ MC38 cells (5×10⁴ cells/well) were incubated with equimolar amounts of anti-CD30 fusion proteins for 1 h on ice. Unbound fusion proteins were either present during the assay or washed out. Freshly isolated NK cells (5×10⁴ cells) were co-incubated for 48 hrs. IL2 (500 U/ml) was added for comparison. NK cell activation was recorded by monitoring IFN-γ secreted into the culture medium. Data represent the mean of triplicates ± standard error of mean (SEM). An experiment out of two with similar results is shown.</p

Figure 5. The HRS3-scFv-IL12-Fc-IL2 dual cytokine fusion protein bound to CD30⁺ cells induces T cell amplification and IFN-γ secretion.

<p>(A, B) Pre-activated T cells were incubated for 48 hrs with serial dilutions of equimolar amounts of the anti-CD30 cytokine fusion proteins or the cytokines IL2, IL12 or IL2 plus IL12. Viable cells were counted; dead cells were excluded by trypan blue exclusion. (C, D) CD30⁺ and CD30⁻ MC38 cells (5×10⁴ cells/well) were incubated with equimolar amounts of anti-CD30 fusion proteins for 1 h on ice. Unbound fusion proteins were either present during the assay (C) or washed out (D). Pre-activated T cells (5×10⁴ cells) were co-incubated for 48 hrs. IL2 (500 U/ml) was added for comparison. T cell activation was recorded by monitoring IFN-γ secreted into the culture medium. Data represent the mean of triplicates ± standard error of mean (SEM). An experiment out of two with similar results is shown.</p