Abstract 568: Click chemistry for theranostic approaches in triple negative breast cancer
Authors: Cristina Simó, Shayla Shmuel, Alex Vanover, Patrícia M. Pereira
Journal: Cancer Research (2025)

Abstract Introduction: Immunotherapies are part of the rapidly expanding landscape of targeted cancer treatments, with market sizes reaching over $150 billion in 2024. However, further understanding of tumor biology is crucial to developing effective therapies that minimize resistance. N-linked glycosylation, a post-translational membrane modification, is well known for its critical role in tumor growth and resistance, its impact on antibody tumor accumulation, and its effect on tumor response to targeted therapies. Despite its significance, methods to image and target (theranostic approaches) N-glycosylation have still not been fully developed. In this study, we use biorthogonal (click) chemistry to visualize and target N-glycans on triple negative breast cancer (TNBC) and exploit them to enhance immunotherapy. Methods: 4T1 TNBC cells were inoculated subcutaneously in Balb/c mice. Glycans visualization: Mice were administered intravenously with tetraacetyl-N-azidoacetylmannosamine (mannose-N3; 40 mg/kg) or PBS (control) once daily for three consecutive days. At 24 h post the last injection, AZDye-680 DBCO (5 mg/kg) was intravenously injected. At 24 h post-AZDye-680 DBCO administration, the organs were harvested, scanned, homogenized, and lysed for fluorescence and protein measurements. Glycans targeting to enhance anti-PD-L1 antibody uptake: Mice were administered mannose-N3 or PBS as described before. Mice were then intravenously injected with the anti-PD-L1 antibody avelumab radiolabeled with copper-64 [64Cu]Cu-NOTA-avelumab-DBCO (6.7 MBq, 40 µg). Mice were imaged with positron emission tomography (PET) at 24 and 48 h. Results: Glycans visualization: 4T1 tumors exhibit high levels of N-glycosylation as visualized through metabolic glycoengineering using a mannose-N3 derivative and AZDye-680 DBCO fluorescent dye. First, mannose-N3 is administered, introducing azide functional groups to cell-surface N-glycoproteins. Next, the AZDye-680 DBCO is administered, clicking with azide groups on the cancer cell’s surface. When compared with PBS, we observed a 3.5-fold increase in tumor uptake in animals administered mannose-N3. Glycans targeting to enhance anti-PD-L1 antibody uptake: Previous studies demonstrated that PD-L1 is highly N-glycosylated in cancer cells, affecting antibody accumulation in tumors. Using immuno-PET with [64Cu]Cu-NOTA-avelumab-DBCO, we showed a 1.9-fold increase in avelumab accumulation in mannose-administered mice compared to PBS controls. Conclusions: In this study, we applied click chemistry to not only visualize N-glycans within tumors but also to exploit their presence to enhance antibody therapy. Our ongoing studies are focused on using metabolic glycoengineering to enhance anti-PD-L1 and anti-PD-1 therapy in TNBC. Citation Format: Cristina Simó, Shayla Shmuel, Alex Vanover, Patrícia M. Pereira. Click chemistry for theranostic approaches in triple negative breast cancer [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 568.