Bispecific antibody (alpha 9-nAChR/mPEG) suppresses angiogenesis and tumor progression of triple-negative breast cancers Tzu-Chun Kan1,2, Chun-Chia Cheng3, Chun-Chun Hsu4,5, Michael Chen6, Jeng-Wei Lu7,8, Sonjid Ochirbat9, Kuo-Hsiang Chuang6, Chien-Chung Chen10, Yi-Yuan Yang11, Yuan-Soon Ho12, Sri Rahayu13, Tzu-Hsuan Huang1,14, Jungshan Chang1,8,15*
1 Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
2 Genomics Research Center, Academia Sinica, Taipei 115201, Taiwan
3 Radiation Biology Research Center, Institute for Radiological Research, Chang Gung University/Chang Gung Memorial Hospital, Taoyuan 33302, Taiwan
4 School of Respiratory Therapy and Graduate Institute of Medical Sciences, College of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
5 Division of Pulmonary Medicine, Department of Internal Medicine, Taipei Medical University Hospital, Taipei 11031, Taiwan
6 Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 11031, Taiwan
7 Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark
8 The Finsen Laboratory, Rigshospitalet/National University Hospital, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
9 International Master/Ph.D. Program in Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
10 Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei 11052, Taiwan
11 School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, New Taipei City 235, Taiwan
12 Institute of Biochemistry and Molecular Biology, College of Life Sciences, China Medical University, Taichung 406040, Taiwan
13 Department of Biology, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
14 Department of Medical Research, Shuang Ho Hospital, Taipei Medical University, New Taipei City 23561, Taiwan
15 International Ph.D. Program for Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan
* To whom correspondence should be addressed. Email: js.chang[at]tmu.edu.tw
Abstract
The α-9 nicotinic acetylcholine receptor (alpha 9-nAChR) has recently emerged as a candidate molecule for targeted therapy of triple-negative breast cancers (TNBCs). To characterize the therapeutic role of alpha 9-nAChR on TNBCs, the alpha 9-nAChR-based bispecific antibody (alpha 9-BsAb) and the alpha 9 BsAb-liposomal doxorubicin conjugates (alpha 9-BsAb/Lipo-Dox) were prepared and assessed. Compared to the group treated with Lipo-Dox alone, a markedly increased nuclear accumulation of alpha 9-BsAb/Lipo-Dox was observed in MDA-MB-231 cells, indicating that alpha 9- nAChR-mediated drug guiding to MDA-MB-231 cells led to enhanced DNA targeting. The therapeutic efficacy of both candidates was assessed in the MTAM (electrospun poly-L-lactic acid microtube array membranes) and TNBC xenografts. The results revealed that the candidates effectively inhibited cell growth within MTAM and tumor growth in TNBC xenografts. Interestingly, a significant reduction in vascular density was observed on MTAM, suggesting that alpha 9-nAChRs play a critical role in angiogenesis and that α-9-BsAb could effectively block this phenomenon. The endothelial cell tube formation assay was conducted to confirm the angiogenic role of alpha 9 nAChRs, revealing a significant reduction in loop numbers in a medium supplemented with alpha 9-BsAb, aligned with the mRNA and protein expression levels observed in tumor samples. These findings indicate that the potential drugs significantly inhibited the expression of angiogenic factors such as VEGF-A, VEGFR2, and phosphorylated VEGFR2 in TNBC xenografts. Moreover, data acquired from disease ontology (DO) and the gene ontology (GO) enrichment analysis in TNBC xenografts exhibited a strongly positive correlation between alpha 9-nAChRs and tumor angiogenesis, cell adhesion, and migration, which was consistent with both in vivo and in vitro angiogenesis studies. Together, alpha 9 nAChR emerges as a promising therapeutic molecule and a potential target for drug d
