<p>Therapeutic cancer vaccines, which induce anti-tumor immunity by targeting specific antigens, constitute a promising approach to cancer therapy. Our previous work developed a novel engineered adenovirus-associated virus 2 (AAV2)-based tumor-specific (neoantigen) cancer vaccine to boost antitumor immunity in combination with radiotherapy, resulting in tumor regression and less distant metastasis. However, the therapeutic efficacy of constitutive vector-based vaccination may be limited by poor immunogenicity, pre-existing vector-specific immunity, and vaccine-induced vector-specific immunity. Here, we examined the combinational therapeutic efficacy of AAV-based cancer vaccine, local radiotherapy, and immune checkpoint blockade (ICB) in different poorly immunogenic cancer animal models. We found that administration with AAV-based neoantigen vaccine significantly increased the response to radiotherapy and ICB, and decreased the risk of distant metastasis. Furthermore, we evaluated a heterologous prime-boost immunization strategy using two optimized AAV serotype vaccines to amplify tumor-specific immunity to neoantigens. These optimized AAV2/AAV6 neoantigen vaccine displayed strong immunogenicity with potent induction of CD8⁺ T cells. As combined with local radiotherapy, the prime-boost vaccine-induced superior tumor clearance and survival compared with other groups. Remarkably, optimized AAV2/AAV6 vaccination promoted CD8⁺ T-cell infiltration in the tumors and elicited the enrichment of T-cell clones. Furthermore, exhausted T-cell marker expression was significantly decreased in the tumor-infiltrating CD8⁺ T cells. Taken together, these results highlight the synergistic potency of engineered AAV2/AAV6 vaccines combined with local radiotherapy for poorly immunogenic cancers.</p>