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Background Arginase-1 (Arg1) contributes to an immune-suppressive tumor microenvironment (TME) and constitutes a significant challenge for cancer immunotherapy. However, clinical interventions to inhibit Arg1 have shown limited success. We reported Arg1-specific memory T cell responses in healthy individuals and cancer patients, 1 and vaccination against Arg1 enhances anti-tumor activity in murine models2 via reducing the immune suppressive function of Arg1+ tumor-associated macrophages (TAMs).3 This study further investigates the mechanism behind Arg1-based peptide vaccination.Methods Anti-tumor activity of Arg1 peptide vaccine was evaluated in 4T1 murine breast cancer model, where Arg1-derived peptide presentation in the TME was assessed by immunopeptidomics. Vaccine-induced immune response was confirmed using an IFNγ ELISpot and TCR sequencing. Tumors were subjected to immunohistochemistry, TCR sequencing, nanostring and flow cytometry. Human CD4+ Arg1-specific T-cell clones were co-cultured with Arg1-expressing TAMs generated in vitro from PBMC-derived CD14+ cells and TAM modulation was evaluated using flow cytometry, Luminex and single-cell RNA sequencing.Results We confirmed Arg1 protein expression and Arg1-derived peptide presentation within the TME of 4T1 tumors through immunohistochemistry and immunopeptidome profiling. Vaccination with Arg1-derived peptides significantly reduced tumor growth, elicited strong immunity and expanded Arg1-specific T cells, detectable through IFNγ and TCR sequencing. Arg1 vaccination led to a significant reduction of Arg1 expression together with genes associated with immunosuppressive TAMs, such as Trem2 and Marco, and myeloid-derived suppressor cells (MDSCs), including S100a8 and S100a9. Moreover, the combination of Arg1 vaccination with anti-PD1 therapy led to an increased anti-tumor effect and distinct gene expression signatures identified through Nanostring. Direct comparison of Arg1 peptide vaccination and Arg1 inhibitor CB-1158 showed that while both treatments led to comparable tumor growth control, Arg1 vaccination promotes a pro-inflammatory TME with significant reduction in Arg1 within TAMs and MDSCs than those observed in mice Arg1 inhibitor-treated mice. Lastly, we established that human Arg1+ TAMs are directly targeted by Arg1-specific T cells, which can reprogram TAMs to increase the expression of co-stimulatory molecules and lead to distinct TAM clustering identified per scRNAseq.Conclusions Immune modulatory vaccine against Arg1 led to anti-tumor activity in a mouse model by shifting the balance from an immunosuppressive to a pro-inflammatory microenvironment through targeting the Arg1+ myeloid compartment, which was not observed with the Arg1 inhibitor CB-1158. Moreover, the anti-tumor activity was further enhanced by combination with anti-PD1. These findings provide a strong rationale for future clinical development of an Arg1-based vaccine as a potential therapeutic strategy for solid tumors.References Martinenaite E, et al. Frequent adaptive immune responses against arginase-1. Oncoimmunology. 2018;7.Aaboe Jorgensen M, et al. Arginase 1-based immune modulatory vaccines induce anticancer immunity and synergize with anti-PD-1 checkpoint blockade. Cancer Immunol Res. 2021;9:1316–1326.Martinenaite E, et al. Arginase-1-specific T cells target and modulate tumor-associated macrophages. J Immunother Cancer. 2025;13.Ethics Approval The animal experiments were reviewed and approved by the Danish Animal Experimentation Council and performed under license number 2022-15-0201-01209. All animal experiments were conducted following national regulations and ethical guidelines.