Reprogramming theTolerogenic Immune Response AgainstPancreatic Cancer Metastases by Lipid Nanoparticles Delivering a STINGAgonist Plus Mutant KRAS mRNA

We demonstrate reprogramming of the tolerogenic immune environment in the liver for mounting an effective immune response against often-fatal pancreatic cancer metastases. This was achieved by engineering a lipid nanoparticle (LNP) to deliver mRNA encoding the KRAS G12D neoantigenic epitope along with cGAMP, a dinucleotide agonist of the stimulator of the interferon genes (STING) pathway, capable of activating a type I interferon response. cGAMP/mKRAS/LNP were synthesized by a microfluidics approach involving nanoprecipitation of mRNA and cGAMP by an ionizable lipid, MC3. Controls included nanoparticles delivering individual components or a wild-type RAS sequence. The dual delivery carrier successfully activated the type I interferon pathway <i>in vitro</i> as well as <i>in vivo</i>, with reprogramming of costimulatory receptor (CD80 and CD86) and MHC-I expression on liver antigen-presenting cells (APC). This allowed the generation of IFN-γ producing cytotoxic T cells, capable of mounting an effective immune response in the metastatic KRAS pancreatic cancer (KPC) mouse model. Noteworthy, intravenous injection of cGAMP/mKRAS/LNP suppressed metastatic growth significantly and prolonged animal survival, both prophylactically and during treatment of established metastases. The protective immune response was mediated by the generation of perforin-releasing CD8⁺ cytotoxic T cells, engaged in pancreatic cancer cell killing. Importantly, the immune response could also be adoptively transferred by injecting splenocytes (containing memory T cells) from treated into nontreated recipient mice. This study demonstrates that reprogramming the immune-protective niche for metastatic pancreatic cancer can be achieved by the delivery of a STING agonist and mutant KRAS mRNA via ionizable LNPs, offering both prophylactic and therapeutic advantages.