In vitro combination therapy of pathologic angiogenesis using anti-vascular endothelial growth factor and anti-neuropilin-1 nanobodies

Document Type : Original Article


Venom and Biotherapeutics Molecules Laboratory, Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran


Objective(s): Emergence of resistant tumor cells to the current therapeutics is the main hindrance in cancer treatment. Combination therapy, which mixes two or more drugs, is a way to overcome resistant problems of cancer cells to current treatments.  Nanobodies are promising tools in cancer therapy due to their high affinity as well as high penetration to tumor sites.
Materials and Methods: Here, the inhibitory effect of mixtures of two nanobodies (anti-vascular endothelial growth factor (VEGF) and anti-neuropilin-1 (NRP-1) nanobodies) on tube formation of human endothelial cells in vitro and ex vivo were analyzed.
Results: Results showed that combination of two drugs significantly inhibited proliferation and tube formation of human endothelial cells. In addition, mixtures of two nanobodies inhibited angiogenesis in chick chorioallantoic membrane (CAM) assay efficiently compared with each individual nanobody.
Conclusion: Results highlight the efficacy of combination therapy of cancer compared with mono-therapy and promises development of novel anti-cancer therapeutics based on nanobodies targeting two or more targets of tumor cells.


1. Roy H, Bhardwaj S, Ylä-Herttuala S. Biology of vascular endothelial growth factors. FEBS lett 2006; 580:2879-2887.
2. Shibuya M. Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases. J Biochem 2013; 153:13-19.
3. Wise LM, Veikkola T, Mercer AA, Savory LJ, Fleming SB, Caesar C, et al. Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1. PNAS 1999; 96:3071-3076.
4. Zachary I, Gliki G. Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res 2001; 49:568-581.
5. Ferrara N, Gerber H-P, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003; 9:669-676.
6. Lampropoulou A, Ruhrberg C. Neuropilin regulation of angiogenesis. Bioch Soc Trans. 2014; 42: 1623-1628.
7. Gelfand MV, Hagan N, Tata A, Oh W-J, Lacoste B, Kang K-T, et al. Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding. Elife 2014; 3: 03720-03739.
8. Lichtenberger BM, Tan PK, Niederleithner H, Ferrara N, Petzelbauer P, Sibilia M. Autocrine VEGF signaling synergizes with EGFR in tumor cells to promote epithelial cancer development. Cell 2010; 140:268-279.
9. Barr MP, Byrne AM, Duffy AM, Condron CM, Devocelle M, Harriott P, et al. A peptide corresponding to the neuropilin-1-binding site on VEGF 165 induces apoptosis of neuropilin-1-expressing breast tumour cells. Brit J Cancer 2005; 92:328-333.
10. Hamerlik P, Lathia JD, Rasmussen R, Wu Q, Bartkova J, Lee M, et al. Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth. J Exp Med 2012; 209:507-520.
11. Pan Q, Chanthery Y, Liang W-C, Stawicki S, Mak J, Rathore N, et al. Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth. Cancer cell 2007; 11:53-67.
12. Kazemi-Lomedasht F, Behdani M, Bagheri KP, Habibi-Anbouhi M, Abolhassani M, Arezumand R, et al. Inhibition of angiogenesis in human endothelial cell using VEGF specific nanobody. Mol Immunol 2015; 65:58-67.
13. Kazemi-Lomedasht F, Muyldermans S, Habibi-Anbouhi M, Behdani M. Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function. Iran J Basic Med Sci 2018; 21:260-266.
14. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005; 438:967-974.
15. Hu C, Jiang X. Role of NRP-1 in VEGF-VEGFR2-independent tumorigenesis. Target Oncol 2016; 11:501-505.
16. Pellet-Many C, Frankel P, Jia H, Zachary I. Neuropilins: structure, function and role in disease. Biochem J 2008; 411:211-226.
17. Rosenfeld PJ, Shapiro H, Tuomi L, Webster M, Elledge J, Blodi B, et al. Characteristics of patients losing vision after 2 years of monthly dosing in the phase III ranibizumab clinical trials. Ophthalmol 2011; 118:523-530.
18. Schmidinger G, Maar N, Bolz M, Scholda C, Schmidt-Erfurth U. Repeated intravitreal bevacizumab (Avastin((R))) treatment of persistent new vessels in proliferative diabetic retinopathy after complete panretinal photocoagulation. Acta Ophthalmol 2011; 89:76-81.
19. Raimondi C, Fantin A, Lampropoulou A, Denti L, Chikh A, Ruhrberg C. Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1-dependent ABL1 activation in endothelial cells. J Exp Med 2014; 211:1167-1183.
20. Prud’homme GJ, Glinka Y. Neuropilins are multifunctional coreceptors involved in tumor initiation, growth, metastasis and immunity. Oncotarget 2012; 3:921-939.
21. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998; 92:735-745.
22. Fuh G, Garcia KC, de Vos AM. The interaction of neuropilin-1 with vascular endothelial growth factor and its receptor flt-1. J Biol Chem 2000; 275:26690-26695.
23. Soker S, Gollamudi-Payne S, Fidder H, Charmahelli H, Klagsbrun M. Inhibition of vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation by a peptide corresponding to the exon 7-encoded domain of VEGF165. J Biol Chem 1997; 272:31582-31588.
24. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 1998; 92:735-745.
25. Cebe-Suarez S, Zehnder-Fjällman A, Ballmer-Hofer K. The role of VEGF receptors in angiogenesis; complex partnerships. Cell Mol Life Sci 2006; 63:601-615.
26. Rajabi M, Mousa SA. The role of angiogenesis in cancer treatment. Biomedicines 2017; 5:34-40.
27.Mokhtari RB, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, Das B, et al. Combination therapy in combating cancer. Oncotarget 2017; 8:38022-38043.
28. Alirahimi E, Ashkiyan A, Kazemi-Lomedasht F, Azadmanesh K, Hosseininejad-Chafi M, Habibi-Anbouhi M, et al. Intrabody targeting vascular endothelial growth factor receptor-2 mediates downregulation of surface localization. Cancer Gene Ther 2017; 24:33-37.
29. Al-Abd AM, Alamoudi AJ, Abdel-Naim AB, Neamatallah TA, Ashour OM. Anti-angiogenic agents for the treatment of solid tumors: potential pathways, therapy and current strategies–a review. J Adv Res 2017; 8:591-605.
30. Zirlik K, Duyster J. Anti-angiogenics: current situation and future perspectives. Oncol Res Treat 2018; 41:166-171.
31. Geretti E, van Meeteren LA, Shimizu A, Dudley AC, Claesson-Welsh L, Klagsbrun M. A mutated soluble neuropilin-2 B domain antagonizes vascular endothelial growth factor bioactivity and inhibits tumor progression. Mol Cancer Res 2010; 8:1063-1073.
32. Bachelder RE, Crago A, Chung J, Wendt MA, Shaw LM, Robinson G, et al. Vascular endothelial growth factor is an autocrine survival factor for neuropilin-expressing breast carcinoma cells. Cancer Res 2001; 61:5736-5740.