Background Siglec-15 is a member of the sialic acid-binding Ig-like lectins (Siglec) family and is a novel target for immunotherapy in cancer. It exhibits low expression in normal tissues but broad expression across cancer indications, specifically on tumor-associated macrophages (TAMs) and tumor cells.¹ To characterize the potential of Siglec-15 as a therapeutic target for solid tumors with high unmet need, an immunohistochemistry (IHC) assay was developed with a proprietary antibody to detect Siglec-15. Using this assay, the expression and prevalence of Siglec-15 was characterized in breast, bladder, cholangiocarcinoma, colon, endometrial, head and neck, kidney, non-small cell lung cancer (NSCLC), and thyroid cancers.

Methods A proprietary monoclonal anti-Siglec-15 antibody was generated from a Siglec-15 positive hybridoma. Antibody binding affinity was assessed by biolayer interferometry (BLI) for Siglec proteins. Western blot analyses were performed to detect Siglec-15 protein in cell lines overexpressing wild type and mutant Siglec-15 at the glycosylation residue N172.² An IHC assay was developed using an automated IHC platform across cell lines and primary human samples. Expression of Siglec-15 was annotated by a board-certified pathologist across ~20 commercial human tumor formalin-fixed, paraffin embedded (FFPE) whole tissue samples from each tumor indication. The average number of Siglec-15 positive macrophages in a 20x microscopic field and the percentage of Siglec-15 positive tumor cells was determined.

Results A proprietary Siglec-15 antibody was able to bind at a high affinity to recombinant Siglec-15 protein with no detectable binding to any other Siglec family protein tested by BLI. The antibody was able to detect Siglec-15 protein by western blot in cell lines overexpressing wild-type and mutant Siglec-15 at N172. This antibody was then used to develop an IHC assay and Siglec-15 was detected in cell lines including those overexpressing Siglec-15 and Siglec-15 N172. Next, the IHC assay was performed on primary human FFPE tumor tissues and Siglec-15 was detected in tumor cells and TAMs. The assay was then validated by evaluating ~20 tumor specimens from each tumor indication for prevalence of Siglec-15. Siglec-15 was observed in TAMs and tumor cells across the tumor indications with NSCLC having the highest positivity rate.

Conclusions A Siglec-15 IHC assay was developed and validated to detect Siglec-15 protein in FFPE tumor tissue samples. The specificity of the assay to detect Siglec-15 on tumor cells and TAMs position it as a potential biomarker assay that may facilitate studies involving Siglec-15 targeting therapeutics.

References

1. Sun J, Lu Q, Sanmamed MF, Wang J. Siglec-15 as an emerging target for next-generation cancer immunotherapy. Clin Cancer Res 2021;27(3):680–688.

2. Wang YL, Wei MB, Zhao WW, Feng LL, Yin XK, Bai SM, Wan XB, Hung MC, Zou AZ, Wang MH, Zheng J, Qin C, Fan XJ. Glycosylation of Siglec15 promotes immunoescape and tumor growth. Am J Cancer Res 2021 May 15;11(5):2291–2302.