Abstract A104: Targeting the CXCR3 axis in HER2+ breast cancer with mRNA Lipid Nanoparticles to enhance anti-tumor immunity

Abstract Breast cancer (BC) is the most frequently diagnosed cancer in women worldwide with more than 2 million new cases in 2020. The efficacy of immune checkpoint blockade (ICB) for BC is limited, with response rates of <10% for anti-PD-1/PDL1 monotherapy in pre-treated triple-negative breast cancer (TNBC) patients and even lower in subtypes that express human epidermal growth factor receptor 2 (HER2+) and/or hormone receptors. Hence, there is an urgent need to improve immune based strategies for BC. One possible approach to enhance response is the application of intra-tumoral (IT) immunotherapy. We have previously reported the efficacy of intratumorally injected plasmid IL-12 (pIL-12-EP) followed by electroporation in a TNBC mouse model. Following pIL-12-EP, treated tumor expressed genes that were associated with increased CD8+ T cell infiltration, activation, trafficking, antigen presentation, and exhaustion, with a striking enrichment of the CXCL9/10/11/CXCR3 family in tumor infiltrating immune cells. This led us to hypothesize that this axis may be the critical component downstream of pIL-12-EP treatment that is responsible for anti-tumor responses in multiple molecular subtypes of BC. In this work, we have gone on to validate the expression of CXCR3 and its ligands by tumor infiltrating lymphocytes (TILs) in biopsies of human HER2+ BC using single cell resolution spatial transcriptomic Xenium instrument. Electroporation has several drawbacks, including insufficient residence time of the plasmid and collateral damage to the surrounding tissue. To overcome these limitations, we used an IT adenoviral injection to treat an implantable HER2+ mouse model. First, we show the sustained expression of our adenoviral encoded target proteins in tumors over several days, a significant increase compared to plasmid or transduced tumor cells. With this treatment, we observed tumor growth inhibition in our Ad-IL12 treated group as compared to the control group with higher CD8 T cell in the treated tumors. Furthermore, Ad-IL12 treated group had higher level of effector memory CD8 T cells, CD8 PD1 cells and higher expression of PD-L1 on myeloid cells along with lower infiltration of neutrophils. Ad-CXCL9 alone was not sufficient to inhibit tumor growth or alter immune cell infiltration. Ongoing efforts to develop a more efficient way of delivering our targeted therapy are focused on lipid nanoparticles (LNPs) containing self-replicating RNA. LNPs have emerged as a well-studied class of delivery systems post COVID-19 and possess a number of advantages over traditional viral vectors or plasmid. These include moderate or less immunogenicity, large payload and simple production with great scalability.1 We conclude that CXCL9 monotherapy has not been effective and propose that a combination or sequential therapy of CXCL9 with IL12 or another immunostimulatory agent like a vaccine may be required. Citation Format: Anchit Bhagat, Jesu Joseph, Joshua D Ginzel, Christopher Rabiola, Amy C Hobeika, Joshua C Snyder, Herbert K Lyerly, Erika J Crosby. Targeting the CXCR3 axis in HER2+ breast cancer with mRNA Lipid Nanoparticles to enhance anti-tumor immunity [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A104.