Abstract
Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, carrying a higher risk of recurrence and metastasis within 3 years, with poor prognosis. Neoadjuvant chemotherapy and immune checkpoint blockade are the standard of care. However, many patients still do not benefit from these treatments. Here we developed an mRNA lipid nanoparticle (LNP) vaccine (Neo20) as a potential therapeutic strategy for TNBC. We aimed to test the preclinical efficacy of Neo20 vaccine in humanized patient-derived xenograft (hu-PDX) mouse models of TNBC expressing HLA class I. Tumor neoantigens were identified in the TNBC PDX model MC1 using a proprietary algorithm that prioritizes vaccine candidates on their likelihood to elicit neoantigen-specific T cells and associated antitumor responses. mRNA encoding 20 HLA-A2-restricted neoantigens was synthesized and encapsulated into LNPs for in vivo administration. Human immunity was reconstituted in female HLA-A2 transgenic NSG mice through I.V. transplantation of CD34+ hematopoietic stem cells (HSCs) from HLA-A2+ cord blood donors. Mice showing efficient human CD45+ (hCD45+) cell engraftment were implanted with MC1 tumors into the mammary fat pad. Once the tumors were established, hu-PDX mice received Neo20 vaccine (10 µg mRNA/mouse) or vehicle/empty LNPs as control by intramuscular (I.M.) injection. Animals were treated every three days for one week, and splenocytes were harvested 7 days after the last dose for restimulation with vaccine peptides followed by IFN-γ ELISpot assay. Flow cytometry analysis of hCD45+ cells showed human reconstitution with T cells, B cells, myeloid cells, and NK cells in blood, spleen, and bone marrow (BM) collected from humanized mice at 12 weeks post-HSCs injection. Within the human T cell fraction, we identified naive, central memory and effector memory CD8+ subsets in the recipient spleen. We also confirmed the ability of T cells to produce IFN-γ, TNF-α, and granzymes A and B, demonstrating their potential to drive effective antitumor responses. Human dendritic cells differentiated into plasmacytoid and conventional subsets in the recipient BM, further supporting this model for the study of acquired immunity post-vaccination. When treated with Neo20 vaccine, hu-PDX mice mounted HLA-A2-restricted immune responses against multiple vaccine peptides as measured by ex vivo IFN-γ production. Luminex analysis of mouse serum demonstrated an increased secretion of proinflammatory cytokines and T cell activation markers in the Neo20-treated group. Overall, our findings provide evidence associated with mRNA vaccines against TNBC and support the use of hu-PDX mice as a model to preclinically validate vaccine candidates. Ongoing studies are directed to evaluate the antitumor response to Neo20 vaccine in hu-PDX mice upon repeated I.M. administration.