AbstractIntroduction:Immune checkpoint inhibitors (ICI) revolutionized the treatment of cancer. However, most patients do not respond to the currently available therapies, or become refractory, which demonstrates the need for the development of novel therapeutic strategies. The transmembrane protein Neuropilin-1 (NRP1) interacts with various receptors and ligands and contributes to an immunosuppressive tumor microenvironment (TME) through multiple mechanisms. NRP1 facilitates the migration of tumor associated macrophages (TAMs), is important for the stability of regulatory T cells, correlates to exhaustion of effector T cells and plays an important role in neoangiogenesis. Furthermore, in several cancers including pancreatic, breast and gastric cancer, high expression of NRP1 has been associated with a poor prognosis. Here we demonstrate the anti-tumor effects of downregulation of NRP1 by locked nucleic acid (LNA) modified antisense oligonucleotides (ASOs) in murine cancer models as monotherapy and in combination with anti-PD-(L)1 antibodies. Furthermore, we provide insights into the potential underlying mechanisms of the observed therapeutic efficacy.Methods:Highly active and well tolerated NRP1-specific ASOs were identified with our OligoCreator oligonucleotide discovery platform. These were tested in different syngeneic tumor models, after a palpable tumor was established, as monotherapy or in combination with ICIs. Survival, immune infiltration, NRP1 expression, and transcriptomic changes were analyzed.Results:A robust knockdown of NRP1 was seen in tumor tissue and in individual infiltrating cell types, including T cells and macrophages, which was accompanied by transcriptomic changes in pathways related to inflammation and extracellular matrix remodeling. Mice treated with NRP1 specific ASO had delayed tumor growth up to complete tumor eradication, which was further increased when combined with ICI, whereas ICI alone showed only a low efficacy. The complete responders were re-challenged with the same cancer cell line, alongside previously untreated mice. While the treatment-naïve mice showed rapid tumor growth, all re-challenged mice eradicated tumors quickly, without the need for an additional treatment.Conclusion:NRP1 specific ASOs are a promising new approach for the treatment of solid cancers. Our data show that the limited efficacy of ICI in the models tested could be improved significantly when combined with the ASO-treatment. Target knockdown was observable in various infiltrating cell types, accompanied by upregulation of genes involved in inflammation and downregulation of genes involved in extracellular matrix organization, which is currently being further analyzed. Based on these promising results, we started preparation for first in human trials to treat patients with solid cancers.Citation Format:Andre Maaske, Sven Michel, Daniel Kokotek, Nicole Kirchhammer, Anne Sadewasser, Julia Festag, Mélanie Buchi, Monika Schell, Janani Sekar, Stefanie Raith, Alfred Zippelius, Frank Jaschinski, Richard Klar. Neuropilin-1 specific antisense oligonucleotides exhibit anti-tumor activity in vivo and significantly increase the efficacy of checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7294.