738 Downregulation of neuropilin-1 expression with antisense oligonucleotides in combination with checkpoint inhibitors demonstrates remarkable anti-tumor activity in vivo

<h3>Background</h3> Unleashing anti-tumor immune responses by immune checkpoint inhibitors is an efficient approach for the treatment of cancer. Still, the majority of patients does not respond to currently available therapies, highlighting the need for the development of novel therapeutic strategies. Neuropilin-1 (NRP1) is a transmembrane protein that interacts with numerous receptors and ligands and contributes to a protumorigenic, immunosuppressive tumor microenvironment (TME). NRP1 supports migration of immunosuppressive macrophages into the TME, maintains the stability of regulatory T cells, contributes to exhaustion of effector T cells and is involved in neoangiogenesis. Accordingly, high expression of NRP1 has been associated with poor survival in several cancers including pancreatic, breast and gastric cancer. Here we show that downregulation of NRP1 using locked nucleic acid (LNA) modified antisense oligonucleotides (ASOs) as monotherapy and in combination with anti-PD-(L)1 antibodies results in strong anti-tumor efficacy in murine cancer models. <h3>Methods</h3> NRP1-specific antisense oligonucleotides were identified using our bioinformatics system Oligofyer™ and ASO discovery platform LNAplus. Different syngeneic tumor cells were implanted into the mammary fat pads of mice. Upon reaching a mean tumor size of ~50–80mm³, mice were randomized and systemically treated with NRP1-specific ASOs +/- anti-PD(L)-1 antibodies. Tumors were excised shortly after treatment initiation for further analysis, or continuously monitored regarding growth and subsequent survival of the mice. Tumors were analyzed via flow cytometry and RNAseq. <h3>Results</h3> Robust knockdown of NRP1 was observed in several intra-tumoral cell types, including macrophages and T-cells already on day 3 after start of treatment. This knockdown led to delayed tumor growth and even complete eradication of tumors in some animals, resulting in a survival benefit in comparison to control animals. Strikingly, we observed a strong on top effect on anti-PD-(L)1 antibodies, leading to complete tumor elimination in around 70% of mice. RNAseq analysis revealed differential expression of genes involved in inflammation, epithelial to mesenchymal transition and other processes in NRP1 ASO treated animals. <h3>Conclusions</h3> The ASO-mediated downregulation of NRP1 is a promising therapeutic strategy that results in potent anti-tumor efficacy as monotherapy and in combination with checkpoint inhibitors. Treatment of mice with NRP1 ASO leads to upregulation of inflammatory genes, potentially contributing to the observed anti-tumor effects, especially in combination with anti-PD-(L)1 antibodies. Ongoing studies aim to fully elucidate the role of NRP1 downregulation in the observed therapeutic efficacy. Encouraged by the promising results, preparation of a clinical trial to treat patients with solid cancers has been started. <h3>Ethics Approval</h3> Approved animal experimental Project No. G-21/113_08E.