*3.3. Comparison with Clinically Relevant Formulations*

We next tested the A-11-LNPs against two clinically relevant RNA-loaded LNP formulations to determine their potential as an mRNA vaccine formulation. The first was the MC3-LNP, also known as Onpattro, a first-ever siRNA therapeutic developed by Alnylam pharmaceuticals for the treatment of transthyretin (TTR) amyloidosis [58,59]. The MC3-LNP was developed as a siRNA formulation targeting liver tissue. The MC3-LNP is considered to be the gold standard LNP formulation and is also widely used as a formulation for mRNA delivery [17,57,60,61]. For these reasons, it was used as a comparative formulation in this study. The second is RNA-LPX, which was reported by Kranz, et al. as a formulation that shows excellent cancer vaccine efficacy by selectively introducing mRNA into splenic DCs [2]. We selected this formulation because it is oriented for the same application as the A-11-LNP, and, because of this, we considered it to be the most suitable control. The MC3-LNP was 59 nm in diameter and showed a 97% mRNA encapsulation. The RNA-LPX was 319 nm in diameter and negatively charged (ζ-potential of −17 mV), consistent with previous reports [2]. The transgene expression level in secondary lymphoid tissues, the spleen and inguinal LN, was next evaluated using Nluc mRNA. The A-11-LNPs showed significantly higher transgene expression levels in both tissues over the two control formulations (Figure 4A). The RNA-LPX showed a superior transgene expression in the spleen compared to MC3-LNPs, which would be reasonable because of the fact that the RNA-LPX was reported as a spleen-selective mRNA delivery system. However, and interestingly, the MC3-LNPs showed a much higher transgene expression in inguinal LN over the RNA-LPX. The transgene expression level in splenic DCs was then measured using EGFP mRNA. The A-11-LNPs achieved approximately 9% of EGFP<sup>+</sup> DCs, which was significantly higher than the two control formulations, indicating the superior transgene expression potency of the A-11-LNPs (Figure 4B). The highest I-A/I-E expression in splenic DCs was also observed for the A-11-LNPs (Figure 4C). These results suggest that the A-11-LNP would have a superior mRNA vaccine effect.

**Figure 4.** Comparison of the A-11-LNPs with clinically relevant formulations. (**A**) Nluc expression in spleen and inguinal LN 24 h after intravenous administration of Nluc-mRNA-loaded formulations at a dose of 0.5 mg mRNA/kg. (**B**) Percentage of EGFP<sup>+</sup> splenic DCs 24 h after the intravenous administration of EGFP-mRNA-loaded formulations at a dose of 0.5 mg mRNA/kg. (**C**) I-A/I-E expression in splenic DCs 24 h after intravenous administration of EGFP-mRNA-loaded formulations at a dose of 0.5 mg mRNA/kg. *n* = 3. \*\* *p* < 0.01.

Motivated by the above results, a prophylactic antitumor experiment in E.G7-OVA tumor-bearing mouse model was conducted. Two sequential administrations of OVA mRNA-loaded A-11-LNPs completely rejected the tumor establishment at all doses tested (Figure 5A). We next examined the therapeutic antitumor effect of OVA mRNA-loaded A-11-LNPs in the same tumor-bearing mouse model. Since a clear shrinkage of tumor tissues was observed at doses of 0.015 mg mRNA/kg or higher in the dose-dependent study (Figure S9), the dose in subsequent experiments was set at 0.03 mg mRNA/kg. A comparative study of the therapeutic antitumor effects of the A-11-LNP and two control formulations (MC3-LNP and RNA-LPX) showed that only the A-11-LNP exhibited significant antitumor activity (Figure 5B). Increased serum IFNβ levels were observed only after the administration of the A-11-LNPs (Figure S10). The A-11-LNPs induced the maturation of splenic DCs, which resulted in the upregulation of the activation markers CD40, CD80, and CD86 (Figure 5C–E). The A-11-LNPs also activated splenic B, T, and natural killer cells, which upregulated the activation marker CD69 (Figure 5F–H). No antitumor activity was observed in the case of the EGFP mRNA-loaded A-11-LNP (Figure 5B), indicating that the antitumor effect depends on the development of an OVA antigen-specific immune response. In addition, B-8-LNPs, which showed significantly lower activity (2.1%) in splenic DCs but exhibited comparable activity (73%) in the whole spleen compared to the A-11-LNPs (Tables S1 and S3), showed no significant antitumor effect (Figure 5B), suggesting that mRNA was efficiently delivered to DCs rather than to the whole spleen, thus leading to antitumor activity. The limited induction of I-A/I-E expression in splenic DCs by the B-8-LNPs also supports this suggestion (Figure S7). Although clear antitumor effects were observed for two doses (0.03 mg mRNA/kg × 2) of the A-11-LNPs, the tumors tended to re-grow after day18 without having completely disappeared (Figure 5B). Based on this observation, the number of doses and dosages were increased (0.125 to 0.5 mg mRNA/kg × 3) to examine the therapeutic antitumor effect. Although there was one individual that experienced a complete response at doses of 0.25 and 0.5 mg mRNA/kg, the results showed a limited increase in the overall antitumor effect (Figure S11). This suggests that the immunosuppressive pathways are enhanced in association with the induction of antitumor immunity by the A-11-LNPs. In fact, E.G7-OVA cells express programmed cell death ligand-1 (PD-L1) [62], one of the major immune checkpoint molecules, and the combination of an adjuvant and an anti-PD-L1 antibody, an immune checkpoint inhibitor (ICI), appears to promote E.G7-OVA tumor shrinkage compared to the use of the adjuvant alone [63,64]. Therefore, the combination of the A-11-LNPs and ICIs would be a reasonable strategy for enhancing an antitumor effect in this tumor model. In addition, when the same tumor cells were re-challenged at 68 days after the initial tumor transplantation in the two individuals that completely responded, they completely rejected tumor engraftment (Figure S12), suggesting that treatment with the A-11-LNPs induced the formation of memory cells, which was also observed for the RNA-LPX in the previous study [2].

Finally, a hematological test was performed after two doses of the OVA mRNA-loaded LNPs at 0.03 mg mRNA/kg had been administered, and no significant alteration in any of the serum chemistry parameters tested was observed, suggesting that the A-11-LNPs are well tolerated under therapeutically relevant doses (Figure 6).

**Figure 5.** Prophylactic and therapeutic antitumor activity. (**A**) Prophylactic antitumor activity of the A-11-LNPs in E.G7-OVA tumor-bearing mice. The OVA mRNA-loaded A-11-LNPs were intravenously injected at the indicated doses twice on 14 and 7 days before tumor inoculation. *n* = 3–4. (**B**) The therapeutic antitumor activity of the A-11-LNPs, MC3-LNP, RNA-LPX, and B-8-LNPs. E.G7-OVA tumor-bearing mice were intravenously injected with OVA mRNA-loaded formulations at two doses of 0.03 mg mRNA/kg on day 8 and 11. *n* = 5. \* *p* < 0.05, \*\* *p* < 0.01. (**C**–**E**) Expression of activation markers CD40 (**C**), CD80 (**D**), and CD86 (**E**) in splenic DCs 24 h after an intravenous injection of OVA mRNA-loaded formulations at a dose of 0.03 mg mRNA/kg. *n* = 3. \* *p* < 0.05, \*\* *p* < 0.01. (**F**–**H**) Expression of an activation marker CD69 in splenic B cells (**F**), T cells (**G**), and NK cells (**H**) 24 h after an intravenous injection of OVA mRNA-loaded formulations at a dose of 0.03 mg mRNA/kg. *n* = 3. \*\* *p* < 0.01.

**Figure 6.** Safety of the A-11-LNPs. Serum chemistry parameters, ALT (**A**), AST (**B**), T-BIL (**C**), LDH (**D**), BUN (**E**), and CRE (**F**) were measured 24 h after the last injection of OVA mRNA-loaded A-11-LNPs at two doses of 0.03 mg mRNA/kg. *n* = 3.
