Abstract 316: Carbohydrate-dependent protein binding antibodies with superior tumor-specificity
Authors: Kehler Patrik , Jaekel Anika , Danielczyk Antje , Goletz Christoph , Lischke Timo , Hartung Evelyn , Weiß Lisa , Weis Manon , Gurka Stephanie , Marinoff Sophie , Kast Naomi , Weiske Manon , Morche Marianne , Gellert Johanna , Neumann Theresa 
Published: June 2022

Abstract
Background: Highly potent therapeutic approaches require very clean targets. However, the majority of antibodies in clinical development or approved for cancer therapy address protein targets that are only overexpressed on cancer cells and yet often show significant expression in healthy organs. Glycans tend to elicit superior tumor specificity as compared to proteins, since glycosylation is strongly altered in cancer, reflecting the drastic changes in tumor metabolism. The changes in glycosylation are mostly due to mutated or mislocated glycosyltransferases and glycosidases, giving rise to highly fucosylated, highly sialylated and truncated glycans. Therefore, proteins expressed in cancer cells can carry tumor-associated carbohydrates like the Thomsen-Friedenreich (TF) and the Thomsen novelle (Tn) antigen as well as their respective sialylated forms sTF and sTn. To increase the tumor-specificity of protein-targeting antibodies, we have developed antibodies against several different protein/carbohydrate combined epitopes (GlycoTargets). Targeting these specific antigens offers reduced on-target/off tumor toxicity, which is key for highly potent therapies.
Methods: The specificity and glycosylation dependency of our antibodies were shown using differentially glycosylated proteins or carbohydrate PAA-conjugates in an ELISA format. Binding to several tumor cell lines and healthy human leukocytes was analyzed via flow cytometry. Binding to different cancer indications as well as related healthy tissues was analyzed by immunohistochemistry.
Results: We have generated antibodies that bind to their target protein only if a specific tumor-associated carbohydrate is present and do not cross-react with the non-glycosylated protein itself, or the carbohydrate structure on different carrier-proteins. Due to the carbohydrate-dependent binding, our antibodies show markedly decreased on-target/off-tumor binding. We will show that our antibodies mediate no binding of different healthy immune cell subsets in comparison to simple protein binding antibodies in flow cytometric analyses. Further, IHC studies reveal that our antibodies stain tumor tissue of different cancer indications but do not stain related normal tissues.
Conclusions: We successfully used the fact that glycosylation is strongly altered in cancer for the generation of highly tumor-specific antibodies that target neither glycan nor protein alone, but the potent combination thereof - a tumor-specific protein/carbohydrate epitope. We could show that by using this approach we were able to reduce on-target/off-tumor binding towards different healthy tissues and healthy immune cell subsets making these antibodies ideal candidates for a therapeutic development.
Citation Format: Patrik Kehler, Anika Jaekel, Antje Danielczyk, Christoph Goletz, Timo Lischke, Evelyn Hartung, Lisa Weiß, Manon Weis, Stephanie Gurka, Sophie Marinoff, Naomi Kast, Manon Weiske, Marianne Morche, Johanna Gellert, Theresa Neumann. Carbohydrate-dependent protein binding antibodies with superior tumor-specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 316.