Intismeran autogene to induce de novo neoantigen-specific T cells as adjuvant therapy in melanoma.

Abstract

9564 Background: Intismeran autogene (intismeran) is an individualized mRNA-based neoantigen (neoAg) therapy designed to enhance endogenous antitumor T-cell responses by targeting patient-specific tumor mutations. In the phase 2 KEYNOTE-942 trial of resected high-risk melanoma, intismeran combined with pembrolizumab (pembro) significantly improved recurrence-free survival (RFS) vs pembro alone. Previously, it was shown that the combination induced greater expansion of de novo T-cell clonotypes, which correlated with RFS in the intismeran arm. Here, we show that de novo T-cell clonotypes are reactive to intismeran-encoded neoAgs. Methods: Peripheral leukapheresis samples longitudinally collected from a subset of intismeran plus pembro treated participants (pts) with melanoma from phase 1 and 2 studies (NCT3313778 and NCT03897881) were assessed at single-cell level to characterize neoAg-specific T cells. In 7 pts, functional assessment of neoAg-reactive CD8⁺ T cells was performed by intracellular cytokine staining and activation-induced marker assays. To define antigen specificity and phenotype of intismeran-induced T cells, we combined longitudinal bulk and single-cell T-cell receptor (TCR) sequencing with functional validation assays from 3 pts. De novo expanded TCRs were tested in Jurkat NFAT-luciferase reporter cells for reactivity against pt-specific intismeran mRNA cassettes and individual neoAgs. For reactive TCRs, minimal epitopes were mapped and mutant vs wild-type peptide reactivity was quantified. Results: Using the combination of bulk and single-cell TCR sequencing and Jurkat NFAT-luciferase reporter assays in 3 pts, we identified 20 TCRs expanded following intismeran therapy across all pts mapping to 11 neoAgs, providing direct evidence of intismeran-driven polyclonal T-cell expansion. These responses targeted multiple neoAgs with persistence >100 days after last intismeran dose. Reactive TCRs showed stronger recognition of mutant vs wild-type peptides, confirming antigen specificity. Functional assessment of T cells from 7 pts showed that neoAg-reactive CD8⁺ T cells secreted IFNγ and TNFα and coexpressed markers for degranulation/activation upon peptide restimulation, demonstrating functional activity. Flow cytometry–based characterization of neoAg-reactive T cells ex vivo revealed that these cells predominantly exhibited an effector-memory phenotype. Upon stimulation, they were polyfunctional (IFNγ⁺ TNFα⁺), mobilized CD107a, and were granzyme B⁺, consistent with cytotoxic effector function relevant to tumor control. Conclusions: Complementary sequencing and functional analyses demonstrated that adjuvant therapy with intismeran plus pembro induced durable, polyclonal, neoAg-specific T-cell responses with effector-memory characteristics, providing mechanistic data consistent with the clinical benefit observed in pts with melanoma. Clinical trial information: NCT3313778 & NCT03897881 .