Neoadjuvant pembrolizumab with therapeutic DNA vaccine reshape intratumoral immunity in a phase 2 trial of HPV-positive HNSCC

Therapeutic cancer vaccines are a promising strategy for inducing antigen-specific T-cell immunity, but their efficacy in solid tumors remains limited by inadequate magnitude, persistence, and functional quality of vaccine-elicited responses. These challenges have intensified efforts to identify adjuvants that can strengthen durable antitumor immunity. Interleukin-7 (IL-7), a cytokine essential for T-cell survival and memory formation, provides a mechanistically grounded strategy for enhancing vaccine responses. To evaluate this in a defined viral antigen setting, HPV-associated head and neck squamous cell carcinoma offers an ideal tumor model for dissecting vaccine immunity and adjuvant mechanisms. We first tested long-acting IL-7 in an HPV16 E6/E7-expressing TC-1 model. IL-7 improved the efficacy of HPV DNA vaccination with PD-1 blockade, producing superior tumor control and enhancing stem-like and memory-associated transcriptional programs within intratumoral CD8⁺ T cells-features consistent with strengthened long-term responsiveness. Guided by these results, we conducted a phase 2 neoadjuvant trial combining pembrolizumab, HPV DNA vaccine (GX-188E), and long-acting IL-7 (GX-I7) in patients with resectable HPV-positive HNSCC. Major pathologic responses occurred in 63.6% of patients, including 36.4% complete responses. Paired single-cell RNA/TCR sequencing and spatial AI analysis revealed coordinated reshaping of the intratumoral immune landscape. A GZMK⁺ effector-memory CD8⁺ population expanded prominently, displaying early memory priming, lymph node-to-tumor recirculation potential, and enrichment of tumor-reactive programs. Increased tertiary lymphoid structures correlated with reduced exhaustion among tumor-reactive CD8⁺ T cells and appeared to facilitate entry and renewal of GZMK⁺ CD8⁺ T cells within tumors. Higher GZMK⁺ CD8⁺ abundance corresponded with greater TCR diversity among tumor-reactive clones, indicating sustained replenishment of antitumor specificities. To reconfirm upstream regulatory pathways, foundation-model-based in silico perturbations showed that knockout of IL7R or STAT5 markedly diminished GZMK-directed differentiation, supporting an IL-7-dependent mechanism underlying this effector-memory programming. Analyses of independent HPV-positive HNSCC cohorts aligned with these observations, showing that tumors enriched for GZMK-programmed CD8⁺ states exhibit stronger antitumor immunity and improved long-term outcomes. Taken together, these comprehensive preclinical, clinical, and exploratory analyses reveal that IL-7 amplifies a GZMK⁺ effector-memory CD8⁺ T-cell program driving sustained recirculation, diversification, and renewal of tumor-reactive CD8⁺ T cells, highlighting a mechanistic axis with potential to enhance the durability of cancer vaccine responses.