Abstract 671: Development of a diagnostic antibody for CD24 targeted therapy

Background: CD24 is a small yet extensively glycosylated glycosylphosphatidylinositol protein that is overexpressed in many tumor cells. Elevated CD24 levels typically indicate a poorer prognosis, as it facilitates malignant activity and confers resistance to chemotherapies. CD24 is also identified as a surface marker of cancer stem cells across multiple tumor types. It triggers an anti-phagocytic by binding to sialic-acid-binding Ig-like lectin 10 (Siglec10) on tumor-associated macrophages, thereby inhibiting the phagocytosis of tumor cells. CD24 has emerged as a promising therapeutic target for anti-cancer treatment. Several clinical trials are being conducted to evaluate the safety and efficacy of CD24-targeted therapies. Here, we developed and characterized an anti-CD24 diagnostic antibody to enhance the screening and selection of patients based on CD24 expression. Methods: SJL mice were immunized with 5-10 million HEK293T-hCD24 cells by intraperitoneal injection. Screening and selection of positive hybridoma clones were conducted using ELISA and flow cytometry analysis. Potential immunohistochemistry (IHC) clones were screened by staining formalin-fixed, paraffin-embedded (FFPE) specimens of human normal esophageal tissue using Leica Bond III platforms. The accuracy assessment, sensitivity (selectivity), specificity, and assay precision were evaluated using the lead antibody. Various human tumor tissue microarrays (TMA), including but not limited to breast cancer, ovarian cancer, urothelial cancer, and small cell lung cancer, were evaluated using lead antibody through IHC staining for further validation. Results: Monoclonal antibody clone ATG1144 binds to the hCD24 core peptide in ELISA with an EC50 of 0.06 nM. Distinct membrane staining on human normal esophageal tissue FFPE specimens can also be observed with IHC staining using ATG1144. For accuracy assessment, six CDX and twenty human specimens, comprising both positive and negative specimens (including solid tumors and B-cell non-Hodgkin lymphomas), were validated. Samples exhibiting high, medium, and low CD24 expression levels were evaluated for sensitivity and specificity, and the interpreted results aligned with the reference outcomes. FFPE tissues from three distinct patients were evaluated for assay precision assessment. The TMA IHC staining result revealed that 50-80% of patients with lung, breast, bladder, ovarian, or liver cancer have CD24 expression on tumor cell surface with low expression in the para-cancerous normal tissue. Conclusion: ATG1144 specifically binds to human CD24 with high sensitivity as demonstrated by IHC staining. The development and validation of the method have been finalized using Leica Bond III platforms. These data suggest a potential diagnostic use of ATG1144 for identifying human CD24 + patients. Citation Format: Peng Chen, Jishun Chen, Min Deng, Rong Guo, Ming Song, Jay Mei, Bing Hou. Development of a diagnostic antibody for CD24 targeted therapy [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 671.