Increasing hydrophobicity in poly(amino acid) adjuvants elicits potent and durable anti-tumor immunity via coordinated cellular and humoral responses.

The coordinated activation of cellular and humoral immunity is essential for robust anti-tumor efficacy and durable immune memory in tumor vaccination. However, most current strategies are biased toward one immune arm, and the rational adjuvant design that efficiently engages both immune arms remains a challenge. Here, the hydrophobic segment content in an amphiphilic poly(amino acid) was optimized to boost both immune responses. The copolymers, poly(L-phenylalanine)m-block-poly(D-lysine)10 (m = 5, 10, or 15; F-DK), were synthesized with identical cationic components and different hydrophobic components. Increasing the phenylalanine ratio induced coordinated changes in nanoparticle size, surface charge, and structural rigidity, thereby enhancing dendritic cell uptake, TLR4-mediated activation, and antigen presentation. After subcutaneous injection into mice, F15-DK10/OVA, with longest chain of poly(L-phenylalanine), triggered robust CD8+ T cell cytotoxicity and strong B cell-mediated antibody response. Meanwhile, splenic central memory T cells and lymph node memory B cells increased by 1.46- and 2.19-fold, compared with the free ovalbumin (OVA). In the OVA-expressing Lewis lung cancer (LLC-OVA) therapeutic model, vaccines with increasing phenylalanine achieved tumor inhibition rates of 31.62%, 65.35%, and 97.81%. Importantly, F15-DK10/OVA achieved 100% tumor-free survival in the LLC-OVA prevention model. Even after two tumor re-challenges, 54.55% of mice remained tumor-free for 644 days, demonstrating durable antigen-specific protection in this model. A CT26 whole-cell-lysate study provided supporting evidence of therapeutic efficacy in one additional syngeneic tumor model. Within the tumor and antigen systems evaluated here, rational tuning of hydrophobic moieties in poly(amino acid) is an effective strategy to enhance tumor vaccine efficacy and long-term anti-tumor immune memory.