Epitope-encoded mRNA-LNP vaccine to enhance anti-tumor potency and persistence of PHOX2B peptide-centric CAR T cells

Background: A Phase 1/1b clinical trial testing peptide-centric chimeric antigen receptor (PC-CAR) T cells that recognize a peptide derived from the major neuroblastoma oncoprotein PHOX2B presented in the context of HLA-A*24:02 and 20 other HLA-A alleles is showing early signs of safety and efficacy (NCT07007117). Anticipated barriers to durable cures include poor persistence and a hostile tumor immune microenvironment (TIME). We hypothesize that persistence and potency can be enhanced, without compromising safety, through the deployment of a “CAR boosting” PHOX2B epitope-encoding mRNA-lipid nanoparticle (LNP) vaccine. Methods: We established a screening platform to test various PHOX2B mRNAs and LNP designs, and here report on the prioritized formulation: a nucleoside-modified PHOX2B 9mer monomer encapsulated by FDA-approved LNP SM-102. In parallel, we established a genetically engineered mouse model (GEMM) of NB to deploy our PHOX2B epitope mRNA-LNP vaccination strategy in vivo . Results: A24 + healthy donor or NB patient monocyte-derived dendritic cells (moDCs) treated with vaccine exhibited log-fold higher PHOX2B 9mer presentation compared to tumor cells and upregulated T cell costimulatory ligands CD80/86 and CD40. PC-CAR T cells exposed to vaccine-treated moDCs had significantly increased proliferation, polyfunctionality, and migratory markers compared to co-cultures with HLA matched NB cell lines. Moreover, vaccine-treated moDCs enriched central, effector, and/or stem cell memory PC-CAR T cell immunophenotypes (donor/patient-dependent) compared to tumor-induced PD1 hi CD39 hi terminal effectors. PC-CAR T cells primed with vaccine-treated moDCs maintained significantly greater cytotoxicity in serial tumor rechallenges. A comprehensive characterization of the immunobiological effects of mRNA-LNP on DC and PC-CAR T cell function using O-link proteomics and transcriptional profiling are ongoing and will be reported. GEMM-derived allografts faithfully recapitulate human disease and express a chimeric HLA-A*24:02/H-2K b MHC that presents a conserved PHOX2B 9mer. Murine (m)PC-CAR T cells engineered with clinical scFv conjoined to murine 41BB- or CD28-CD3ζ were polyfunctional and cytotoxic against A24/H-2K b allografts. Vaccine-treated A24/H-2K b knock-in mice presented PHOX2B 9mer by DCs in spleen and lymph nodes, activating PC-CAR T cells. Comprehensive in vivo evaluation of mPC-CAR-T expansion, memory formation, and anti-tumor potency with TIME spatialomic profiling are ongoing and will be reported. Conclusion: These IND-enabling studies will inform our clinical vaccine dosing strategy with the recommended Phase 2 PC-CAR T cell dose in an upcoming trial amendment. This CAR boosting vaccine may not only improve efficacy of PC-CAR T cells for NB but also guide mRNA-LNP enhancement strategies for other adoptive cellular therapies.