mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables effificient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-ofexperiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specifific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentifified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profifile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarififies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNALNP-based antitumor immunotherapy.
INTRODUCTION
Messenger RNA (mRNA) vaccines are extremely versatile, as mRNA sequences can be easily tailored to encode any antigen of interest, enabling both rapid and mass-scale vaccine development against emerging pathogens as well as personalized vaccine design against cancers. Key to the success of the currently approved SARS-CoV2 mRNA vaccines and of many mRNA-based prophylactic vaccines in clinical development are lipid-based nanoparticles (LNPs), delivery vehicles that mediate effificient mRNA expression in situ and endow the vaccine with intrinsic adjuvant properties.
LNPs are composed of an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid, with the ionizable lipid being considered the most important driver of mRNA expression. In contrast to their predecessors having a cationic charge across a wide variety of pH ranges, ionizable lipids are pH-sensitive lipids that endow the LNPs with a positive, membrane fusion-promoting charge at endosomal pH while having a neutral charge at physiological pH. The ionizable lipid is required to encapsulate the mRNA and drives endosomal escape. The PEGylated lipid improves LNP stability and controls the interaction of
the LNPs with blood proteins and cells. Cholesterol and the phospholipid contribute to LNP stability and endosomal membrane destabilization.
Intramuscular or subcutaneous administration of mRNA LNPs has been reported to elicit CD8 T cell responses against the mRNA encoded antigen. In these strategies, mRNA is delivered to local antigen-presenting cells (APCs), resulting in low to moderate antigen-specifific CD8 T cell levels in the circulation. However, to effectively combat tumors (especially at distant locations) potent, long-lasting, and systemic CD8 T cell responses are required. Intravenous (i.v.) delivery of mRNA vaccines has been described to confer enhanced T cell responses and antitumor immunity, which is likely related to its capacity to mobilize the large pools of APCs present in the spleen. Induction of strong CD8 T cell responses has been confifirmed clinically, upon i.v. immunization of metastatic melanoma patients with mRNA lipoplexes composed of 1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE) and the cationic lipid DOTMA, but no LNP-based formulation has yet been used in the clinic for i.v. therapeutic cancer vaccination. LNPs might show even further improved activity compared with lipoplexes given their enhanced intrinsic potential to transfect cells. Nonetheless, standard LNP formulations typically deliver RNA to hepatocytes upon i.v. delivery, instead of the APCs needed to instigate CD8 T cells. In this study, we speculated that LNPs could be tailored toward systemic vaccination through optimization of the molar ratio between the four lipids and selection of an appropriate polyethylene glycol (PEG) lipid. These parameters are known to impact LNP features such as size, z potential, pharmacokinetics, and biodistribution, which ultimately will determine in vivo immunogenicity.
To design and screen a virtually unlimited variety of potential LNP compositions in a timeand cost-effective manner, we adopted a quality-by-design approach based on a statistical design-of-experiment (DOE) coupled to Bayesian regression modeling. This strategy enabled us to identify the key composition parameters that determine immunogenicity and to predict optimal LNP compositions within a single round of in vivo immunogenicity assessment. Optimal LNP compositions
(within the constraints of the design space) resulted in strong CD8 T cell responses that could be boosted by repeated administration and conveyed antitumor effificacy in a syngeneic mouse TC-1 tumor model. Mechanistic studies revealed that the T cell response was dependent on initial mRNA expression by various APCs in the spleen and on the induction of type I interferons (IFNs). Surprisingly, we also discovered a previously unrecognized role of B cells in instigating antigen-specifific CD8 T cell responses upon intravenous mRNA-LNP vaccination.
Bevers et al., mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenicimmune cells, Molecular Therapy (2022), https://doi.org/10.1016/j.ymthe.2022.07.007
