A BCMA-mRNA vaccine is a promising therapeutic for multiple myeloma

Abstract

Cancer vaccines are emerging as promising therapies to not only prevent cancer but to treat cancer. Here, we developed a therapeutic vaccine for multiple myeloma (MM) using B-cell maturation antigen (BCMA) protein as a target. Given the remarkable efficacy of COVID-19 messenger RNA (mRNA) vaccines, we first packaged sequence- and base-optimized BCMA mRNA into lipid nanoparticles (LNPs) using next-generation ionizable lipid, enhancing their accumulation in the spleen. A toll-like receptor 3 agonist, polyinosinic:polycytidylic acid [poly(I:C)], was also encapsulated in LNPs to further elicit BCMA-specific immune response. BCMA-mRNA LNPs were internalized by dendritic cells (DCs) in vitro, triggering proliferation and activation of BCMA-specific CD8+ cytolytic T cells (CTLs). Importantly, these CTLs lysed BCMA+ U266 MM cells and CD138+ patient MM cells, without affecting BCMA-knockout U266 or CD138- patient-derived bone marrow cells. Vaccination of C57BL/6J mice with BCMA-mRNA LNPs activated splenic DCs and induced BCMA-specific CTLs, assessed by tetramer staining, which selectively killed murine 5TGM1 BCMA overexpressing MM cells. Finally, vaccination of C57BL/KaLwRijHsd mice bearing BCMA-overexpressing 5TGM1 cells inhibited tumor growth associated with BCMA-specific CD8+ T-cell responses. The combination treatment with poly(I:C) further triggered the immune response induced by BCMA-mRNA LNPs in all instances. Our findings provide the framework for clinical evaluation of BCMA-mRNA LNP vaccines to improve patient outcome in MM.

Graphical abstract

Figure 1.

Synthesis and characterization of mRNA LNPs. (A) The different lipid components used for the packaging of BCMA mRNA and poly(I:C) in LNPs. (B-C) The cryogenic electron microscopy images of the BCMA-mRNA LNPs and poly(I:C) LNPs. (D-E) Particle size analysis performed for BCMA LNPs (82.3 ± 6.75 nm) and poly(I:C) LNPs (117.6 ± 5.86 nm) by dynamic light scattering. (F) The loading percentage of BCMA-mRNA LNPs and poly(I:C) LNPs. (G) The gel electrophoresis shows the stability of free mRNA and mRNA packaged in LNPs after incubation in human serum for 2 hours at 37°C. (H) The switch in surface charge from negative at physiological pH 7.4 to positive at pH 5, mimicking the acidic endosomal environment, confirmed the efficient endosomal escape of the mRNA into cytosol. DOPE, 1-2-dioleoyl-sn-glycero-3-phosphoethanolamine.

Figure 2.

Transfection and translation efficacy of mRNA LNPs in MoDCs. (A-D) MoDCs were treated with free Cy5-tagged BCMA mRNA, Cy5-tagged BCMA mRNA packaged in LNPs, or Cy5-BCMA-mRNA LNPs codelivered with poly(I:C) LNPs. They were evaluated for cellular uptake of Cy5-tagged BCMA-mRNA LNPs using flow cytometry after 1 hour and 2 hours of incubation, and cytosolic localization (red signal) was evaluated by confocal microscopy after 1 hour. Quantification was performed for respective treatment groups using flow cytometry and confocal microscopy. (E-H) MoDCs were treated with free GFP mRNA, GFP-encoding mRNA packaged in LNPs, or GFP-encoding mRNA LNPs codelivered with poly(I:C) LNPs. MoDC uptake of GFP was analyzed by flow cytometry after 12 hours and 24 hours of incubation, and cytosolic expression was evaluated by confocal microscopy after 24 hours of incubation. GFP quantification was determined for respective treatment groups using flow cytometry and confocal microscopy. The data are shown as mean ± standard deviation (SD) from at least 3 independent biological experiments. Statistical analysis was performed using an unpaired Student t test: ∗P < .05. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3.

Evaluation of in vitro immune response in primary human T cells and cytotoxic effect of CTLs on cancer cells. (A) Isolation of T cells and MoDCs from the peripheral blood of the same healthy donor. After treatment, MoDCs were cocultured with T cells for 5 days. (B) DC activation marker CD80 was analyzed on CD11c⁺ MoDCs after treatment with different hBCMA-mRNA cohorts along with irrelevant mRNA or mock-loaded LNP. (C-D) For the T-cell proliferation assay, naïve T cells were prestained with CFSE and cocultured for 5 days with pulsed MoDCs; then the percentage of CFSE-stained CD8⁺ T cells and CD4⁺ T cells was determined by flow cytometry. (E-F) The hBCMA-specific CD8⁺ T-cell percentage was assessed by hBCMA-specific tetramer staining using flow cytometry. The quantification of hBCMA-specific CD8⁺ T-cell percentage was determined after treatment with different hBCMA-mRNA LNPs, irrelevant mRNA, or mock-loaded LNP. (G-J) ELISpot analysis was performed with the treated MoDCs and T cells coculture to quantify the spot-forming CD8⁺ T cells for both TNF-α and IFN-γ, dual staining of the same well treated with control and hBCMA-mRNA cohorts. (K-L) CTLs were generated from the 5-day coculture of CD8⁺ T cells and treated MoDCs from the patient peripheral blood, and their cytotoxicity was evaluated against same patient bone marrow-derived CD138⁺ MM cells. For flow analysis, CD138⁺ MM cells were first gated with CD138-APC–positive population followed by MM cell death percentage evaluated by PI staining with respective quantifications. (M-N) CTLs generated from the 5-day cocultures of CD8⁺ T cells and treated MoDCs isolated from the peripheral blood of healthy donors and the cytotoxicity of CTLs were evaluated against bone marrow–derived CD138⁺ MM cells and CD138⁻ stromal cells from patients with MM. (O) The cytotoxicity of these isolated CTLs was determined against CFSE-stained U266-WT and U266-BMCA-KO cell lines. The data are shown as mean ± SD from 3 independent experiments. Statistical analysis was performed using an unpaired Student t test: ∗P < .05. IFN-γ, interferon gamma; PI, propidium iodide; SFC, spot-forming cells; SSC, side scatter; TNF-α, tumor necrosis factor α.

Figure 4.

In vivo distribution of LNPs in major organs and evaluation of in vivo stability and translation efficacy of its packaged mRNA. (A-B) Cellular uptake of DiR-loaded LNPs by DCs isolated from the liver, spleen, and lung of C57BL/6J mice 6 hours, 12 hours, and 24 hours after IV and IM injection. (C-E) The free F-Luc mRNA and F-Luc-mRNA loaded in LNPs were injected into C57BL/6J mice by the IV route to monitor the in vivo stability of mRNA and translation efficacy. Shown is the representative real-time live imaging of animals 6 hours after injection along with quantification of BLI for different treatment groups. (F-G) The in vivo tissue distribution of DiR-loaded LNPs was monitored in the liver, with quantification of liver accumulation of DiR dye at different time points, 6 hours, 12 hours, and 24 hours after IV administration of DiR-loaded LNPs. 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. BLI, bioluminescence imaging; DAPI, 4′,6-diamidino-2-phenylindole; DiR, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide.

Figure 5.

Evaluation of in vivo DC activation after IV and IM injection of mRNA LNPs. (A-B) C57BL/6J mice were treated with free mBCMA mRNA or mRNA LNPs with or without poly(I:C) LNPs by either the IV or IM route. After 24 hours of treatment, the spleens were isolated and splenocytes were analyzed for DC activation markers CD80 or CD40 on CD11c⁺ DCs. (C-E) Immunofluorescence analysis of the spleen showing activation of CD11c (yellow) and CD80 (magenta) after 24-hour treatment with different groups. The scatter plots represent percentage of CD80⁺ DCs after IV and IM injections of different treatment groups. 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. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 6.

Analyzing BCMA-specific tetramer⁺ CD8⁺ T cells and infiltration of T cells in splenic tissue. C57BL/6J mice received different treatment groups by either IV or IM injection. Five days after a single IV dose, and 7 days after the first IM dose in a schedule of 2 doses separated by 3 days, the mice were euthanized, and their splenocytes were stained to assess the BCMA-specific tetramer⁺ CD8⁺ T cells by flow cytometry. (A-B) Percentage of BCMA-specific tetramer–positive cells analyzed on splenocytes after IV and IM injections in different treatment groups. (C) The spleens were further analyzed for infiltrating CD3⁺ and CD8⁺ T cells by immunofluorescence staining. The representative images demonstrate the infiltrating CD3⁺ (green) and CD8⁺ (yellow) T cells in splenic tissue. (D-E) The percentage of CD8⁺ T cells infiltrating into the spleen after different treatments is shown after IV and IM injection. 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. DAPI, 4′,6-diamidino-2-phenylindole.

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.