Abstract by Jinjin Li

Breast cancer (BC) remains the most prevalent malignancy among women, with triple-negative breast cancer (TNBC) constituting 15-20% of all cases. TNBC is associated with poor prognosis, aggressive progression, and limited treatment options. Cancer vaccines based on messenger RNA (mRNA) represent a promising strategy for eliciting antigen-specific immune responses. As a mammary gland-specific protein, α-lactalbumin (α-LA) is aberrantly overexpressed in approximately 70% of TNBC tumors, and it has emerged as a relevant tumor-associated antigen for vaccination. This study aimed to investigate lipid-based mRNA vaccines targeting α-LA for TNBC immunotherapy. Specifically, the roles of ionizable lipids and helper lipids in modulating mRNA delivery efficiency and immunostimulatory properties were investigated. Lipid nanoparticles (LNPs) and lipid-polymer hybrid nanoparticles (LPNs) were formulated with ionizable lipids, i.e., L5N12, C12-200, and SM-102, and with helper lipids including cholesterol and the immunopotentiating synthetic analog monomycoloyl glycerol (MMG-1). These formulations were characterized with respect to physicochemical properties and evaluated in vitro for transfection efficiency, cytotoxicity, and immune activation, including dendritic cell maturation and pattern-recognition receptor signaling. Additionally, in vivo immunogenicity profiles of α-LA-encoding mRNA vaccines were assessed, and their antitumor efficacy was evaluated in a murine 4T1 TNBC model.

L5N12-modified nanocarriers demonstrated Toll-like receptor 4 (TLR4) activation in human but not murine reporter cells and promoted dendritic cell activation. C12-200-based LNPs exhibited efficient mRNA transfection and intrinsic adjuvant effects but induced substantial cytotoxicity. They induced robust dendritic cell maturation in vitro, mediated via human stimulator of interferon genes (STING) but not TLR4 pathways. Substituting cholesterol with MMG-1 in C12-200-based LNPs reduced both transfection efficiency and immune activation. In contrast, SM-102-based LNPs mediated high transfection efficiency with minimal toxicity, and MMG-1 substitution further enhanced antigen expression, suggesting lipid-specific synergy. Intramuscular immunization of mice with SM-102/cholesterol LNPs elicited α-LA-specific CD8⁺ T-cell responses. However, prophylactic vaccination failed to suppress tumor growth in vivo, indicating that vaccine-induced immune activation may be insufficient for tumor control in an aggressive 4T1 TNBC model. In conclusion, rational design of lipid-based mRNA vaccines enables fine-tuning of immunogenicity and delivery efficiency. While ionizable lipids such as L5N12 and C12-200 provide adjuvant potential, biocompatibility remains a challenge. SM-102-based formulations demonstrate a favorable balance, offering a promising platform for TNBC vaccination. These findings support continued development of self-adjuvanting mRNA vaccines with optimized lipid compositions for effective cancer immunotherapy.