Abstract LB211: Development of novel immune checkpoint inhibitor: Anti-siglec-9 antibody

Abstract Current immune checkpoint inhibitor (CPI) therapies targeting PD-1 and CTLA4 pathways have revolutionized cancer treatment, offering durable responses in some patients. However, their efficacy can be limited to a subset of individuals, and challenges such as resistance and immune-related adverse events remain significant. Recent studies on siglecs (sialic acid-binding immunoglobulin-like lectins) have suggested their important roles in tumor immunosurveillance including driving immunosuppression in the TME. In a recent study, we reported that targeting siglec-7 by a novel antibody strongly enhanced natural killer cells (NKs) mediated killing in ovarian cancer treatment. Siglec-9 is another emerging immune checkpoint molecule. The interaction between siglec-9 and its ligands is associated with poor prognosis in HCC cancer and enhanced immune suppression in prostate cancer and GBM. Siglec-9 is also upregulated in melanoma tumor-infiltrating CD8+ T cells. The presence of siglec-9 ligands inhibited the cytotoxic function of CD8+ T cells. The development of effective tools to interrupt this pathway is important. In this study, we detected siglec-9 ligands expression on human cancer cell lines by using recombinant siglec-9 protein through flow. We found that siglec-9 ligands were highly expressed in multiple cancer cells, including melanoma, ovarian cancer, gastric cancer, breast cancer, pancreatic cancer and EBV+ nasopharyngeal carcinoma. To interrupt the interaction between siglec-9 and the ligands, we generated a library of anti-siglec-9 monoclonal antibodies (mAbs). We immunized mice with gene encoded human siglec-9 extracellular domain (ECD) and used 10x genomics sequencing to analyze antibody sequences and developed 100 synthetic human antibodies for analysis based on frequences and binding and in depth studied 9 clones. Clones were tested in ELISA, Western blot and SPR against siglec-9 ECD then further analyzed by flow against siglec-9 transduced 293T cells, which resulted in 7 dominate and 2 weak binding clones. Using antibody competition analysis by flow, we observed that none of these 7 clones were competing for binding suggesting unique epitope reactivities. We analyzed the binding of the 9 siglec-9 mAbs against different immune cell populations in human PBMCs. We found that all9 clones showed binding to CD56dimCD16+ NKs, CD56dimCD16- NKs, CD56brightCD16- NKs, NKT, cDC and monocytes, but to different extents. By incubation of siglec-9 mAbs with human PBMCs for 24 hours, 5/9 clones significantly downregulated the expression of siglec-9 on the NKs subset, an important phenotype for further study. In competitive analysis, one clone efficiently blocked siglec-9 protein binding to all the siglec-9 ligands which were expressed on melanoma cell lines. These siglec-9 mAbs represent important new tools for detection of siglec-9 on immune cells, and as potential therapeutic agents to block the suppressive function of siglec-9 ligand binding. These humanized mAbs offer unique tools to potentially enhance existing immunotherapy strategies. Citation Format: Shushu Zhao, Devivasha Bordoloi, Candice Ionescu, Joshua Jose, Pratik S. Bhojnagarwala, Nicholas Tursi, David B. Weiner. Development of novel immune checkpoint inhibitor: Anti-siglec-9 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB211.