Fig. 5: Sequencing of APOBEC3B^ACTIVE modified vaccines generates reproducible mutations in CSDE1.

Sanger sequencing of CSDE1 from a parental B16 cells, b APOBEC3B^ACTIVE modified vaccine, and c APOBEC3B^INACTIVE modified vaccine was performed. Figures are representative of three independent experiments. Each preparation of the APOBEC3B^ACTIVE modified vaccine had proportions of cells containing a C or a T at the 13th base pair, corresponding to the P5S amino acid change seen in Fig. 5 and Supplementary Fig. 2. (Figure was prepared using SnapGene software (from GSL Biotech; available at snapgene.com). d On day 0, 2 × 10⁵ B16 murine melanoma cells were implanted subcutaneously into the right flank of C57Bl/6 mice. Two 5-day courses of B16-APOBEC3B^ACTIVE, B16-APOBEC3B^INACTIVE, B16-CSDE1, or B16-CSDE1^* vaccines (freeze/thaw lysate of 1 × 10⁶ cells i.p.) were administered from days 5 to 9 and 12 to 16. This was followed by anti-PD1 antibody or IgG control (12.5 mg/kg i.p.) on days 12–16, 19, 21, and 23. Kaplan–Meier survival curves representing experiment described. Representative of three separate experiments. e On day 0, 5 × 10⁴ B16 murine melanoma cells were implanted into the brainstem of C57Bl/6 mice. One 5-day course of B16-APOBEC3B^ACTIVE, B16-APOBEC3B^INACTIVE, B16-CSDE1, or B16-CSDE1^*-modified cell vaccines (freeze/thaw lysate of 10⁶ cells i.p.) was administered from days 5 to 9. This was followed by anti-PD1 antibody or IgG control (12.5 mg/kg i.p.) on days 12, 14, 16, 19, 21, and 23. Kaplan–Meier survival curves representing experiment described (n = 7 mice/ group). This experiment was repeated once. f Spleens and lymph nodes obtained from mice treated with B16-CSDE1 or B16-CSDE1^* vaccination and antibody-mediated checkpoint inhibition in (e) were made into single-cell suspensions and cocultured with B16 or TC2 target cells expressing CSDE1 or CSDE1^* for 72 h. Supernatant from the coculture was assayed using a mouse interferon gamma ELISA. Data points represent three biologic replicates. Error bars indicate mean and SD. ANOVA was used followed by Tukey’s multiple comparison test. **p ≤ 0.01, ***p ≤ 0.001.