From left: Dr Yang Yuansheng, Dr Tan Heng Liang, Dr Simeon Cua, Dr Andre Choo, Dr Vanessa Ding, Fong Wey Jia, Angela Chin and Ally Lau
Simeon Cua1, Heng Liang Tan1, Wey Jia Fong1, Angela Chin1, Ally Lau2, Vanessa Ding1, Zhiwei Song3, Yuansheng Yang4 and Andre Choo1, 5
1 Stem Cells 1 Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore
2 Proteomics Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore
3 Expression Engineering 1 Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore
4 Animal Cell Technology 1 Group, Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore
5 Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore
Published in Oncotarget 2017 9(17): 13206-13221 (Online Version)
As targeted drugs, monoclonal antibodies (mAbs) and antibody drug conjugates (ADCs) can bind to surface antigens that are selectively expressed or overexpressed on cancer cells. Even so, a limited number of mAb and ADC treatment options currently exist and an even fewer number of antigen targets have been exploited (eg. HER2, EGFR, CD20). To treat the diverse spectrum of cancer including different molecular subtypes, the identification of new antigens and the development of therapeutic mAbs or ADCs against these targets are needed. Oncofetal antigens represent one such type (eg. CEA, AFP). These antigens are found in cancer but are also transiently expressed during normal embryonic development1. To identify new therapeutic mAb candidates against oncofetal antigens, our group generated multiple panels of novel mAbs against surface markers on hESC and screened them against cancer cells. Two of these mAbs targeted known oncofetal proteins, the epithelial cell adhesion molecule (EpCAM) and the podocalyxin-like protein 1 (PODXL)2,3.
Here, we report on the characterization and development of another antibody, mAb 2448. Interestingly, the antigen target of mAb 2448 was identified as an N-glycosylated form of Annexin A2 (ANXA2), a calcium dependent phospholipid binding protein. Screening data revealed that mAb 2448 binds not only to the surface of hESC but also a wide range of carcinomas including ovarian and breast cancer (Figure 1). Selectivity in both indications was to an epithelial (E) or intermediate epithelial phenotype (IE) that was classified according to the Epithelial-Mesenchymal Transition (EMT), a critical process during cancer progression. To demonstrate that mAb 2448 is a good therapeutic candidate, a chimeric variant (ch2448) was evaluated using various in vitro functional assays and mouse xenograft models. Two potential mechanisms of actions were reported. First, naked antibody ch2448 elicited strong ADCC activity which was further enhanced by afucosylation. Second, ch2448 demonstrated efficient internalization and as an ADC, was used for delivery of the plant-derived toxin, saporin. Taken together, these results indicate that embryonic ANXA2 is an attractive therapeutic target in ovarian and breast cancer and suggest that ch2448 can be further developed as a naked mAb or ADC therapy.
1. Brewer, B. G., Mitchell, R. A., Harandi, A. & Eaton, J. W. Embryonic vaccines against cancer: an early history. Exp. Mol. Pathol. 86, 192–197 (2009).
2. Ng, V. Y., Ang, S. N., Chan, J. X. & Choo, A. B. H. Characterization of Epithelial Cell Adhesion Molecule as a Surface Marker on Undifferentiated Human Embryonic Stem Cells. STEM CELLS 28, 29–35 (2010).
3. Choo, A. B. et al. Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells Dayt. Ohio 26, 1454–1463 (2008).
Figure 1. Binding of Antibody 2448 on the Surface of Ovarian and Breast Cell lines. Antibody 2448 was screened against live cells by flow cytometry. Grading was done according to binding percentages after marker gating with a 2.0-2.5% cut-off from the negative control: “-” < 10% binding, “+” = 10-40% binding, “++” = 40-60%, “+++” = 60-80% and “++++” > 80%.
†“E” = Epithelial, “IE” = Intermediate Epithelial, “IM” = Intermediate Mesenchymal, “M” = Mesenchymal and “U” = Not Classified. Cell lines 184B5, HCC1395 and IOSE523 were not classified (U).