Optimized lentiviral backbone induces robust and diverse T cell immunity against neoantigens to counteract tumor heterogeneity.

Targeting personalized tumor neoantigens is a promising strategy in immuno-oncotherapy, tumor heterogeneity requires that adaptive immunity be effectively induced against a broad spectrum of neoantigens for a significant anti-tumor effect. We developed several non-integrative lentiviral vectors encoding optimized immunogen that induce robust T cell responses against neoepitopes derived from murine colorectal tumors. Incorporating proteasome-targeting sequences and 4-alanine spacers between neoepitopes enhanced responses to both dominant and subdominant neoepitopes. Using longer natural sequences encompassing minimal epitopes further improved immunogenicity. These vectors drove complete regression of MC38 tumors expressing the selected neoepitopes and sustained immune memory to prevent relapse. Additionally, these vectors synergized with anti-programmed cell death protein 1 (PD1) therapy to inhibit wild-type MC38 tumor growth. Variant allele frequency tracking demonstrated T cells eradicated neoantigen-positive cells without affecting negative ones. Our results validate lentiviral vectors for personalized neoepitope therapy and underscore the need for diverse neoantigens in immunotherapy against tumor mosaicism.