Additional file 1 of Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice

Additional file 1: Sup. Fig. 1. (A) ELISA showing binding of V-aCD3Mu (Coupled)(Kd = 38.8, Bmax = 3.31), rVAR2 (Kd and Bmax not determined), and V-aCD3Mu (Fused)(Kd = 14.2, Bmax = 3.36) to CSPG on a decorin backbone. Data is representative of a minimum of two separate experiments. (B) Solid 4T1 tumors 50-100 mm3 in size were treated with either PBS (n=5), V-aCD3Mu (Coupled) + CpG (n=8), or V-aCD3Mu (Fused) + CpG (n=8) on day 10, 12, 14, and 17 after tumor injection. Numbers in parentheses indicate the number of animals with complete tumor regression out of all mice in the group. Sup. Fig. 2. (A) Gating strategy on splenocytes and PBMCs in flow cytometry used to determine binding of rVAR2, aCD3Mu, V-aCD3Mu, aCD3Hu, and anti-V5 antibodies to T cells and non-T cell splenocytes/PBMCs. The gating is single cells lymphocytes live cells CD4+ and/or CD8+ cells as T cells and CD4-CD8- cells as non-T cells. The geometric MFI of the anti-penta-HIS antibodies conjugated to Alexa Flour 488 was then used to evaluate the binding of the HIS-tagged proteins. (B) Binding of aCD3Mu (Kd = 4.96, Bmax = 1.05), rVAR2 (Kd = NR, Bmax = 0.46), and V-aCD3Mu (Kd = 1.24, Bmax = 3.38) to murine recombinant CD3 in ELISA with aCD4Mu as a negative control (left). Means and standard deviations are shown. Right pane shows CSA inhibition of binding at 120 nM (right). Each dot represents one data point. Sup. Fig. 3. Cytokines measured from 4T1 and splenocyte co-culture supernatants using ELISA. Mouse splenocytes were incubated with 4T1 cancer cells together with 200 nM of the indicated protein. Sup. Fig. 4. (A) Survival curves for mice with indicated tumors treated as described in Fig. 4. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05. (B) Bioluminescence in vivo imaging of C57BL/6 mice following orthotopic implantation of 5x104 Luciferase+ primary pancreatic cancer cells (CHX2000) derived from KPC mice (LSL-KrasG12D/+; p53f/f; Pdx1-Cre). Sup. Fig. 5. (A-C) Survival curves for mice treated as described in Fig. 5. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Sup. Fig. 6. (A) Treatment schedule until day 14 when spleens and tumors were harvested for flow cytometry and the subsequent gating strategy on splenocytes to evaluate different cell types in C-D. (B) Percentage of live cells relative to the PBS group in the spleen. Both CD8+ and CD4+ T cells that are CD69+, CD44hi, CD8+CD25+, or Tregs are shown. (C) UMAPs of splenocytes from all four treatment groups with clustering performed in ClusterExplorer. Cell types in clusters are explained below. Statistics were performed using one-way ANOVA with Dunnett’s post hoc test for comparison of all treatment groups to the PBS group. P values are indicated if significant or important for reading the figure. (D) Correlations between the tumor size and �8+CD69+ (p=0.67) and �4+CD69+ (p=0.14) of all live single cells in the tumor evaluated by simple linear regression. Sup. Fig. 7. Binding of mouse antibodies to 4T1 cells and B16-F10 cells in flow cytometry. Serum from C57BL/6 mice treated as described in materials and methods was diluted as illustrated on the figure and incubated with 200.000 4T1 or B16-F10 cells. Soluble CSA was added if indicated for 1 hour before detection with an anti-mouse IgG antibody conjugated to FITC.

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