Engineering A Biomaterials-based Lymphoid Niche for mRNA Lipid Nanoparticle Cancer Vaccines

Introduction: Immunotherapy has witnessed a paradigm shift recently with the advent of mRNA LNPs as an accelerated approach to combat cancer. The success of the two mRNA vaccines has demonstrated the effectiveness of stabilized mRNA constructs to deliver the encoded antigen(s) and the safety and potency of LNPs in delivering the gene therapeutics and vaccines to relevant cells in vivo. Nonetheless, realizing the full potential of mRNA LNP cancer vaccines requires further optimization of LNP immune activation profiles and delivery strategies to permit more efficient programming of immune responses for anticancer efficacy. Herein, we propose an off-the-shelf mRNA LNP-loaded nanofiber-hydrogel matrix (LiNx), which promotes endogenous immunocyte infiltration and creates a programmable immunostimulating microenvironment, therefore maximizing antigen expression and presentation (Fig. 1a). Previously, we analyzed 1,080 LNPs to boost transfection efficiency in antigen-presenting cells (APC)[1]. Our findings indicate that precise tuning of LNP composition enables the modification of transgene expression in both APCs and non-APCs, thereby influencing the immune activation profile[1]. Here, we prepared LiNx formulations with three LNP vaccines screened from the library and examined the effect of the microgel niche on cell recruitment activity and transfection efficiency among different immune cell types. We then investigated antigen presentation, immune cell activation in LiNx, and antigen-specific immune response. More importantly, we assessed the efficacy of a single immunization with LiNx in comparison to the conventional three-dose prime-and-boost regimen for free LNPs, and the synergistic effects of combining the optimized mRNA LiNx cancer vaccine with systemic immune checkpoint blockade in therapeutic cancer models.

Materials and

Methods: DLin-MC3-DMA was purchased from MedKoo Biosciences. DOPE, DSPC, 18PG, and DMG-PEG-2000 were from Avanti Polar Lipids. Cholesterol was from Sigma-Aldrich. All mRNA (Cre mRNA, OVA mRNA) molecules were from TriLink BioTechnologies. The anti-CTLA-4 (α-CTLA-4) monoclonal antibody (mctla4-mab10-10) was from InvivoGen. Sodium hyaluronate (HA) with molecular weight of 1.5 × 10^6 Da was from LifeCore Biomedical Inc. Glycidyl acrylate was from TCI America Inc. Poly(ethylene glycol) dithiol with an average molecular weight of 5 kDa (PEG-SH, MW 5 kDa) was from JenKem Technology. All other chemical reagents were purchased from Sigma Aldrich unless otherwise noted.
To prepare LiNx, the NHC microgel (G′ of ~250 Pa) was combined with LNPs using the syringe extrusion method [2]. The kinetics of host cell recruitment was examined following injection of LiNx on C57BL/6 mice (30 μg mOVA per mouse, s.c.). To assess cell transfection in vivo, Ai9 mice were s.c. administered with LiNx containing 30 μg mCre per mouse. For tumor inhibition studies, mice aged 6–8 weeks were injected with MC38-OVA or B16-OVA cells (1 × 10^6 in prophylactic and 3 × 10^5 in therapeutic studies, s.c.) into the right flank. Tumor growth was monitored every two days. In therapeutic tumor studies, vaccinations began when tumor sizes were less than 50 mm^3 (around day 4 after tumor inoculation). Animals were immunized by s.c. injection of different LNPs or LiNx formulations containing mOVA. One dosage of LiNx were given and a total of three doses were given for free LNP group.

Results, Conclusions, and Discussions: Results
To investigate host cell recruitment in LiNx, we injected mOVA LiNx into mice and harvested composites at 3- and 7-days. Incorporating D6 LNPs significantly increased host immune cell recruitment compared to LiNx itself (Fig. 1b). A substantial number of APCs, particularly macrophages, were detected on day 3 (Fig. 1c). We further assessed the delivery efficacy of LiNx using Cre mRNA with Ai9 mice and found that D6 LiNx resulted in substantial tdTom+ cell including macrophages and DCs in composites (Fig. 1d). The anti-tumor effect of D6 LiNx was investigated in MC38-OVA and B6-OVA therapeutic tumor models (Fig. 1e). D6 LiNx demonstrated potent tumor suppression, resulting in a median survival time of 75 days with 50% of mice remaining tumor-free beyond 100 days (Fig. 1f). Notably, D6 LiNx outperformed free LNPs given in a standard immunization schedule with three doses (median survival time of 37.5 days), emphasizing the superior efficacy of this single-dose hydrogel vaccine. Additionally, D6 LiNx was assessed in a prophylactic melanoma model (Fig. 1g), showing stronger tumor growth inhibition and a prolonged median survival time of 30 days (Fig. 1h).

Conclusion
We present an effective approach to potentiate mRNA LNP cancer vaccine by creating an immune cell recruiting and retention microgel matrix as a local immunostimulating niche. The LiNx matrix does not rely on lymph node trafficking of LNPs, but rather leverages its capability to locally enhance antigen expression and presentation, as well as immune cell crosstalk, resulting in potent antigen-specific responses. Among the three LiNx formulations evaluated, D6 LiNx demonstrated sustained host cell recruitment and differentiation, showcasing superior antitumor efficacy compared to both other LiNx formulations and the traditional three-dose regimen for D6 LNP vaccination. These findings underscore the potential of this strategy to enhance immune responses triggered by mRNA LNPs through biomaterials-based lymphoid niche modulation. Our work presents a versatile vaccine delivery platform with broad applicability in various disease treatment and prevention strategies, thereby extending the scope of mRNA LNP immunotherapies.

Reference
[1] Zhu Y, et al., Nat. Biomed. Eng. 2023 Dec. doi: 10.1038/s41551-023-01131-0.
[2] Yao Z-C, et al., Small. 2022 Aug. doi: 10.1002/smll.202202309.

Acknowledgements (Optional): Acknowledgements

Funding: This study is partially supported by National Institutes of Health grants U01AI155313 (H.-Q.M.) and P41EB028239 (H.Q.M. and J.P.S.).
Author contributions: Y.Z. and H.-Q.M. conceived of and designed this study. H.-Q.M. and J.P.S. secured the funding for this study. Y.Z., J.M., Z.-C.Y., S. L., J.L.S., J.L., X.L., and C.W. performed the experiments. Y.Z., S.K.R., J.P.S., J.C.D., J.W.H. and H.-Q.M. participated in data analysis and interpretation. The abstract was written by Y.Z. and H.-Q.M., with revisions and inputs from all the other authors.