Supplementary Figures S1-S10 from A Novel Antibody–IL15 Fusion Protein Selectively Localizes to Tumors, Synergizes with TNF-based Immunocytokine, and Inhibits Metastasis

Abstract

<p>S1 shows the biochemical characterization by SDS-PAGE and size-exclusion chromatography of: A. IL15-F8-F8, B. F8(Db)-IL15, C. F8-F8-SD, D. KSF-KSF-IL15, E. KSF-KSF-SD-IL15, F. L19-L19-IL15 and G. F8(scFv)-SD-IL15. S2 shows the biochemical characterization and comparison with cartoon, size-exclusion chromatography and CTLL2 activity assay of: A. F8-F8-SD-IL15, B. F8-F8-IL15-SD, C. SD-IL15-F8-F8 and D. IL15-SD-F8-F8. n.p. = not performed. S3 shows the amino acid sequence of F8-F8-IL15 (starting from N-terminus: F8 VH, F8 VL, IL15) and F8-F8-SD- IL15 (starting from N-terminus: F8 VH, F8 VL, Sushi Domain, IL15) and the fusion protein production and purification protocol. S4 shows the biological activity of F8-F8-IL15 and F8-F8-SD-IL15 was tested with a NF-kB reporter cell line that secretes luciferase upon activation of NF-kB pathway by cytokines as IL15. S5 shows the microscopic fluorescence analysis of EDA expression on F9, C51 and WEHI-164 tumor sections detected with F8-F8-IL15 and F8-F8-SD-IL15 (green) and anti-CD31 (red). KSF-KSF-IL15 and KSF- KSF-SD-IL15 were used as negative control. 10x magnification, white scale bar = 100 �m. S6 shows the quantitative biodistribution analysis of radioiodinated L19-L19-IL15 in immunocompetent mice bearing F9 teratocarcinoma tumors. Results are expressed as percentage of injected dose per gram of tissue (3 mice). S7 shows body weight changes in experimental animals A. Toxicity expressed as % of body weight loss in mice treated 3 times i.v. into the later vein with 0.6 μg/g, 1 μg/g and 1.5 μg/g of F8-F8-SD-IL15. The dose of 1.5 μg/g was found to be lethal for all mice after the third injection. B. Toxicity expressed as % of body weight loss in lung metastasis mice model injected with C51 tumor cells. C. Toxicity expressed as % of body weight loss in mice treated 3 times i.v. into the later vein with 0.6 μg/g of F8-F8-SD-IL15 plus 0.1 μg/g of F8mTNF. S8 shows body weight changes in experimental animals A. Toxicity expressed as % of body weight loss in mice treated 3 times i.v. into the later vein with saline control group, anti-PD1 antibody (10 μg/g), F8-F8-IL15 (5 μg/g) alone (black arrows) or in combination with anti-PD1 antibody (10 μg/g) (grey arrows); F8-F8-SD-IL15 (0.6 μg/g) alone (black arrows) or in combination with anti-PD1 antibody (10 μg/g) (grey arrows). B. Toxicity expressed as % of body weight loss in mice treated 3 times i.v. into the later vein with saline control group, F8mTNF (0.1 μg/g), anti-PD1 antibody (10 μg/g), F8-F8-IL15 (5 μg/g) alone or in combination with anti-PD1 antibody (10 μg/g) and F8mTNF (0.1 μg/g); F8-F8-SD-IL15 (0.6 μg/g) alone or in combination with anti-PD1 antibody (10 μg/g). In one group F8-F8-SD-IL15 was administered at (0.3 μg/g) in combination with F8mTNF (0.1 μg/g). S9 shows FACS gating strategies. A. Gating strategy used to analyse tumor draining lymph nodes composition. The fluorescent-minus one (FMO) control is inserted in order to gate the AH1-specific CD8+ T cells. B. Gating strategy used to analyse FoxP3 positive CD4+Tcells. C. Gating strategy used to analyse tumor composition. D. Gating strategy used to analyse specific AH1+CD8+Tcells in tumors. The fluorescence-minus one (FMO) control is inserted in order to gate the AH1-specific CD8+ T cells. E. Gating strategy used for pBMC characterization for CD4+ , CD8+ and NKp46+ . F. Gating strategy used for pBMC characterization for FoxP3+ . S10 shows analysis by flow cytometry of blood taken after 6 hours from mice i.v. injected with PBS (100μl), F8- F8-IL15 (5 μg/g) and F8-F8-SD-IL15 (0.6 μg/g). Different levels of CD4, CD8, APC and NK cells were observed upon different treatments.</p>