Supplementary Data from Targeting Tumor Vasculature with TNF Leads Effector T Cells to the Tumor and Enhances Therapeutic Efficacy of Immune Checkpoint Blockers in Combination with Adoptive Cell Therapy
Authors: Elia Angela Rita , Grioni Matteo , Basso Veronica , Curnis Flavio , Freschi Massimo , Corti Angelo , Mondino Anna , Bellone Matteo 
Published: April 2023

<p>5 supplementary figures Figure S1. ICB does not prolong survival of TRAMP mice. A) Kaplan-Meier survival curve of 16-18 week-old TRAMP mice untreated (Untreated; dotted line; n=14), or treated with isotype specific antibodies (IgG; gray line; n=5), or ICB (green line; n=5). B) Sixteen-18 week old TRAMP mice received ICB and NGR-TNF. The urogenital organs were recovered at day 40. Coded sections were analyzed by H&E and scored according to signs of complete regression (CR; black area), partial regression (PR; gray area), or no regression (NR; white area). The pie graph shows that all mice (n=3) were scored as NR. Figure S2. Phenotype and functional activity of ACT-derived CD8+ T cells infiltrating melanomas. Mice bearing a 7-day-old B16-OVA melanoma were randomly assigned to the treatments described in Fig. 2. Mice were killed when the mean tumor diameter reached approximately 4 mm. Single cell suspensions from each tumor were analyzed by flow cytometry. The panels show representative contour plots of cells gated on ACT-derived CD3+ CD8+ Vβ5.1+ Vα2+ cells, and analysed for expression of CD44 and CD62L (A), CD44 and PD1 (B), Granzyme B (C) and IFN-γ production (D). Data are representative of two independent experiments Figure S3. Phenotype and functional activity of endogenous CD8+ T cells infiltrating melanomas. Mice bearing a 7-day-old B16-OVA melanoma were randomly assigned to the treatments described in Fig. 2. Mice were killed when the mean tumor diameter reached approximately 4 mm. Single cell suspensions from each tumor were analyzed by flow cytometry. The panels show representative contour plots of cells gated on endogenous CD3+ CD8+ Vβ5.1- Vα2- cells, and analysed for expression of CD44 and CD62L (A), CD44 and PD-1 (B), Granzyme B (C) and IFN-γ production (D). Data are representative of two independent experiments. Figure S4. NGR-TNF improves efficacy of ICB by recruiting endogenous T cells into protective anti-tumor immune responses. Tumors from mice treated as described in the legend to Fig. 3 were processed to single cells and analyzed by flow cytometry. Graphs report percentage (mean {plus minus} SEM;) of OT1 cells and endogenous CD8+ T cell within CD8+ T cells (A, F respectively), and percentages (mean {plus minus} SEM) of CD44+ CD62L- cells (B and G), CD44+ PD-1+ cells (C and H), Granzyme B+ cells (D and I), and IFN-γ + cells (E and L) within the OT1 (A-E) or the endogenous CD8+ T cell subpopulations (F-L). Each symbol represents an individual mouse. Data are aggregated from two independent experiments. Student t-test: * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P < 0.0001; ns, not significant. Figure S5. Therapeutic efficacy of the combination between ICB and NGR-TNF in MC38- bearing mice. A) Schematic representation of the experimental treatment. C57BL/6 mice were subcutaneously challenged with MC38 colon cancer cells (5 x105 /mouse; kindly provided by M.P.Colombo, Milan, Italy). Before injection, Mycoplasma infection was excluded by PCR. When the tumor reached 3 mm in mean diameter, mice were randomly assigned to either one of following treatments: vehicle (PBS; n=9), IgG+NGR-TNF (n=8), anti-CTLA-4 (100 µg/mouse) and anti-PD-1 (250 µg/mouse) antibodies (ICB n=12), or ICB+NGR-TNF (n=12). Mice were followed therefore for tumor growth and survival. B-D) Tumor volume of individual mice treated with PBS (B), IgG+NGR-TNF (C), ICB (D), or ICB+NGR-TNF (E). Each line represents one mouse. F) KaplanMeir plot reporting the survival of the mice treated as described in A. Log-rank test: * P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P < 0.0001; ns, not significant.</p>