Figure 7.
Specific cytotoxicity of in vivo CTLs on murine tumor cells and effect of therapeutic vaccination on tumor growth and immune responses in the C57BL/KaLwRijHsd murine model. (A-C) The spleens were isolated from treated C57BL/6J mice 5 days after a single IV dose, and 7 days after the first IM dose in a schedule of 2 doses separated by 3 days, for different treatment groups. CD8+ T cells were isolated from splenocytes and cultured with CFSE prestained 5TGM1-WT and 5TGM1-BCMA-OE cells to evaluate cytotoxicity. After 24 hours of incubation, cell death percentage was evaluated by PI using flow cytometry. (D) Experimental workflow for in vivo therapeutic vaccination. C57BL/KaLwRijHsd mice were subcutaneously inoculated with 6 × 106 5TGM1-BCMA-OE cells and randomized into 4 treatment groups once the tumor reached 60 to 80 mm3. Mice received different mBCMA-mRNA LNPs twice weekly for 2 weeks by IV injections. The spleens were removed for analysis 5 days after the final treatment. (E) A significant reduction in tumor volume was observed in groups treated with BCMA-mRNA LNPs and BCMA-mRNA+poly(I:C) LNP, compared with control and free mRNA. (F) The treatment with mBCMA-mRNA+poly(I:C) LNP selectively inhibited growth of 5TGM1-BCMA-OE but not 5TGM1-WT tumors. (G-I) After treatment, splenocytes were isolated and stained for flow cytometry to assess the BCMA-specific tetramer positivity on CD8+ T cells. The representative plots and scatter plots demonstrate increased BCMA-specific tetramer+ CD8+ T cells in treated groups. (J) mRNA LNP vaccine’s mechanism of action. LNPs can efficiently deliver mRNA to antigen-presenting cells, such as DCs. Activated DCs can present processed antigens to educate T cells for efficient priming and amplification of T cells specific to MM cells. Cytotoxic T cells specifically target BCMA-expressing tumor cells or CD138+ MM cells but do not target cells without BCMA surface expression. The data are shown as mean ± SD from at least 3 independent biological experiments. Statistical analysis was performed using an unpaired Student t test: ∗P < .05. PI, propidium iodide.

Specific cytotoxicity of in vivo CTLs on murine tumor cells and effect of therapeutic vaccination on tumor growth and immune responses in the C57BL/KaLwRijHsd murine model. (A-C) The spleens were isolated from treated C57BL/6J mice 5 days after a single IV dose, and 7 days after the first IM dose in a schedule of 2 doses separated by 3 days, for different treatment groups. CD8+ T cells were isolated from splenocytes and cultured with CFSE prestained 5TGM1-WT and 5TGM1-BCMA-OE cells to evaluate cytotoxicity. After 24 hours of incubation, cell death percentage was evaluated by PI using flow cytometry. (D) Experimental workflow for in vivo therapeutic vaccination. C57BL/KaLwRijHsd mice were subcutaneously inoculated with 6 × 106 5TGM1-BCMA-OE cells and randomized into 4 treatment groups once the tumor reached 60 to 80 mm3. Mice received different mBCMA-mRNA LNPs twice weekly for 2 weeks by IV injections. The spleens were removed for analysis 5 days after the final treatment. (E) A significant reduction in tumor volume was observed in groups treated with BCMA-mRNA LNPs and BCMA-mRNA+poly(I:C) LNP, compared with control and free mRNA. (F) The treatment with mBCMA-mRNA+poly(I:C) LNP selectively inhibited growth of 5TGM1-BCMA-OE but not 5TGM1-WT tumors. (G-I) After treatment, splenocytes were isolated and stained for flow cytometry to assess the BCMA-specific tetramer positivity on CD8+ T cells. The representative plots and scatter plots demonstrate increased BCMA-specific tetramer+ CD8+ T cells in treated groups. (J) mRNA LNP vaccine’s mechanism of action. LNPs can efficiently deliver mRNA to antigen-presenting cells, such as DCs. Activated DCs can present processed antigens to educate T cells for efficient priming and amplification of T cells specific to MM cells. Cytotoxic T cells specifically target BCMA-expressing tumor cells or CD138+ MM cells but do not target cells without BCMA surface expression. The data are shown as mean ± SD from at least 3 independent biological experiments. Statistical analysis was performed using an unpaired Student t test: ∗P < .05. PI, propidium iodide.

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