Original articles
Meng Li, Hanmei Xu, Junzhi Wang. Optimized functional and structural design of dual-target LMRAP, a bifunctional fusion protein with a 25-amino-acid antitumor peptide and GnRH Fc fragment[J]. Acta Pharmaceutica Sinica B, 2020, 10(2): 262-275

Optimized functional and structural design of dual-target LMRAP, a bifunctional fusion protein with a 25-amino-acid antitumor peptide and GnRH Fc fragment
Meng Lia,b, Hanmei Xuc, Junzhi Wanga,b
a Shenyang Pharmaceutical University, Shenyang 110016, China;
b Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National Institutes for Food and Drug Control, Beijing 102629, China;
c State Key Laboratory of Natural Medicines, Ministry of Education, the Engineering Research Center of Synthetic Polypeptide Drug Discovery and Evaluation of Jiangsu Province, Department of Marine Pharmacy, China Pharmaceutical University, Nanjing 211198, China
To develop fusion protein of a GnRH Fc fragment and the integrin targeting AP25 antitumor peptide for GnRH receptor-expressing cancer therapy. The LMRAP fusion protein was constructed. A transwell invasion assay was performed. The gene mRNA and protein levels of GnRHR-I, α5β1, and αvβ3 in different cancer cell lines were assessed. Cell proliferation was measured using a cell counting kit-8. An antagonist assay was performed on GnRH receptors. Anti-tumor activity was evaluated with a mouse xenograft tumor model. Immunohistochemistry (IHC) was applied to detect CD31 and CD34 expressions. Pharmacokinetic characteristics were determined with an indirect competition ELISA. The developed bifunctional fusion protein LMRAP not only inhibited HUVEC invasion, but also inhibited proliferation of GnRHR-I, α5β1, and αvβ3 high expression cancer cells. The IC50 for LMRAP in the GnRH receptor was 6.235×10-4 mol/L. LMRAP significantly inhibited human prostate cancer cell line 22RV1 proliferation in vivo and in vitro. LMRAP significantly inhibited CD31 and CD34 expressions. The elimination half-life of the fusion protein LMRAP was 33 h in rats. The fusion protein made of a GnRH Fc fragment and the integrin targeting AP25 peptide retained the bifunctional biological activity of GnRHR blocking, angiogenesis inhibition, prolonged half-life and good tolerance.
Key words:    Fusion protein    GnRH    Integrin    Angiogenesis    Prostate cancer   
Received: 2019-06-22     Revised: 2019-09-19
DOI: 10.1016/j.apsb.2019.10.010
Funds: The subject of the National Science and Technology Major Projects for Major New Drugs Innovation and Development supported this study (grant No. 2018ZX09736016-007, China).
Corresponding author: Junzhi Wang     Email:wangjz_nifdc2014@163.com
Author description:
PDF(KB) Free
Meng Li
Hanmei Xu
Junzhi Wang

1. Stanislaus D, Pinter JH, Janovick JA, Conn PM. Mechanisms mediating multiple physiological responses to gonadotropin-releasing hormone. Mol Cell Endocrinol 1998;144:1-10.
2. Bono AV, Salvadore M, Celato N. Gonadotropin-releasing hormone receptors in prostate tissue. Anal Quant Cytol Histol 2002;24:221-7.
3. Halmos G, Arencibia JM, Schally AV, Davis R, Bostwick DG. High incidence of receptors for luteinizing hormone-releasing hormone (LHRH) and LHRH receptor gene expression in human prostate cancers. J Urol 2000;163:623-9.
4. Imai A, Tamaya T. GnRH receptor and apoptotic signaling. Vitam Horm 2000;59:1-33.
5. Emons G, Grundker C, Gunthert AR, Westphalen S, Kavanagh J, Verschraegen C. GnRH antagonists in the treatment of gynecological and breast cancers. Endocr Relat Cancer 2003;10:291-9.
6. Silversides DW, Murphy BD, Misra V, Mapletoft RJ. Monoclonal antibodies against LHRH:development and immunoactivity in vivo and in vitro. J Reprod Immunol 1985;7:171-84.
7. Xu J, Zhu Z, Wu J, Liu W, Shen X, Zhang Y, et al. Immunization with a recombinant GnRH vaccine conjugated to heat shock protein 65 inhibits tumor growth in orthotopic prostate cancer mouse model. Cancer Lett 2008;259:240-50.
8. Fromme B, Eftekhari P, Van Regenmortel M, Hoebeke J, Katz A, Millar R. A novel retro-inverso gonadotropin-releasing hormone (GnRH) immunogen elicits antibodies that neutralize the activity of native GnRH. Endocrinology 2003;144:3262-9.
9. Hsu CT, Ting CY, Ting CJ, Chen TY, Lin CP, Whang-Peng J, et al. Vaccination against gonadotropin-releasing hormone (GnRH) using toxin receptor-binding domain-conjugated GnRH repeats. Cancer Res 2000;60:3701-5.
10. Ferro VA, Stimson WH. Investigation into suitable carrier molecules for use in an anti-gonadotrophin releasing hormone vaccine. Vaccine 1998;16:1095-102.
11. Yin R, Zheng H, Xi T, Xu HM. Effect of RGD-4C position is more important than disulfide bonds on antiangiogenic activity of RGD-4C modified endostatin derived synthetic polypeptide. Bioconjug Chem 2010;21:1142-7.
12. Zhao HL, Yao XQ, Xue C, Wang Y, Xiong XH, Liu ZM. Increasing the homogeneity, stability and activity of human serum albumin and interferon-α2b fusion protein by linker engineering. Protein Expr Purif 2008;61:73-7.
13. Amet N, Lee HF, Shen WC. Insertion of the designed helical linker led to increased expression of TF-based fusion proteins. Pharm Res 2009; 26:523-8.
14. Bai Y, Ann DK, Shen WC. Recombinant granulocyte colonystimulating factor-transferrin fusion protein as an oral myelopoietic agent. Proc Natl Acad Sci U S A 2005;102:7292-6.
15. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal sample preparation method for proteome analysis. Nat Methods 2009;6:359-62.
16. Leutert M, Menzel S, Braren R, Rissiek B, Hopp AK, Nowak K, et al. Proteomic characterization of the heart and skeletal muscle reveals widespread arginine ADP-ribosylation by the ARTC1 ectoenzyme. Cell Rep 2018;24:1916-29. e5.
17. Cox J, Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 2008;26:1367-72.
18. Mann K, Edsinger E. The Lottia gigantea shell matrix proteome:reanalysis including MaxQuant iBAQ quantitation and phosphoproteome analysis. Proteome Sci 2014;12:28.
19. Ciechanowska M, Kowalczyk M, Lapot M, Malewski T, Antkowiak B, Brytan M, et al. Effect of corticotropin releasing hormone and corticotropin releasing hormone nist on biosynthesis of gonadotropin releasing hormone and gonadotropin releasing hormone receptor in the hypothalamic-pituitary unit of follicular-phase ewes and contribution of kisspeptin. J Physiol Pharmacol 2018;69:451-61.
20. Liu K, Tian T, Zheng Y, Zhou L, Dai C, Wang M, et al. Scutellarin inhibits proliferation and invasion of hepatocellular carcinoma cells via down-regulation of JAK2/STAT3 pathway. J Cell Mol Med 2019; 23:3040-4.
21. Ramos-Alvarez I, Jensen RT. P21-activated kinase 4 in pancreatic acinar cells is activated by numerous gastrointestinal hormones/neurotransmitters and growth factors by novel signaling, and its activation stimulates secretory/growth cascades. Am J Physiol Gastrointest Liver Physiol 2018;315:G302-17.
22. Fidanzi-Dugas C, Liagre B, Chemin G, Perraud A, Carrion C, Couquet CY, et al. Analysis of the in vitro and in vivo effects of photodynamic therapy on prostate cancer by using new photosensitizers, protoporphyrin IX-polyamine derivatives. Biochim Biophys Acta Gen Subj 2017;1861:1676-90.
23. Bjornsdottir I, Sternebring O, Kappers WA, Selvig H, Korno HT, Kristensen JB, et al. Pharmacokinetics, tissue distribution and excretion of 40 kDa PEG and PEGylated rFVIII (N8-GP) in rats. Eur J Pharm Sci 2016;87:58-68.
24. Robinson S, Delongeas JL, Donald E, Dreher D, Festag M, Kervyn S, et al. A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development. Regul Toxicol Pharmacol 2008;50:345-52.
25. Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester RGS, Barzi A, et al. Colorectal cancer statistics, 2017. CA Cancer J Clin 2017;67:177-93.
26. Zhou M, Li L, Li L, Lin X, Wang F, Li Q, et al. Overcoming chemotherapy resistance via simultaneous drug-efflux circumvention and mitochondrial targeting. Acta Pharm Sin B 2019;9:615-25.
27. Dlugi AM, Miller JD, Knittle J. Lupron depot (leuprolide acetate for depot suspension) in the treatment of endometriosis:a randomized, placebo-controlled, double-blind study. Lupron Study Group. Fertil Steril 1990;54:419-27.
28. Lebret T, Culine S, Davin JL, Hennequin C, Mignard JP, Moreau JL, et al. Quality of life of 1276 elderly patients with prostate cancer, starting treatment with a gonadotropin-releasing hormone agonist:results of a French observational study. Aging Male 2014;17:87-93.
29. Chang JI, Bucci J. Unusual side effect from a luteinizing hormonereleasing hormone agonist, leuprorelin, in the treatment of prostate cancer:a case report. J Med Case Rep 2016;10:323.
30. Finstad CL, Wang CY, Kowalski J, Zhang M, Li ML, Li XM, et al. Synthetic luteinizing hormone releasing hormone (LHRH) vaccine for effective androgen deprivation and its application to prostate cancer immunotherapy. Vaccine 2004;22:1300-13.
31. Etlinger HM, Gillessen D, Lahm HW, Matile H, Schonfeld HJ, Trzeciak A. Use of prior vaccinations for the development of new vaccines. Science 1990;249:423-5.
32. Schutze MP, Leclerc C, Jolivet M, Audibert F, Chedid L. Carrierinduced epitopic suppression, a major issue for future synthetic vaccines. J Immunol 1985;135:2319-22.
33. Sad S, Chauhan VS, Arunan K, Raghupathy R. Synthetic gonadotrophin-releasing hormone (GnRH) vaccines incorporating GnRH and synthetic T-helper epitopes. Vaccine 1993;11:1145-50.
34. Zuazo-Gaztelu I, Casanovas O. Unraveling the role of angiogenesis in cancer ecosystems. Front Oncol 2018;8:248.
35. Cui C, Merritt R, Fu L, Pan Z. Targeting calcium signaling in cancer therapy. Acta Pharm Sin B 2017;7:3-17.
36. Li Y, Cozzi PJ. Angiogenesis as a strategic target for prostate cancer therapy. Med Res Rev 2010;30:23-66.
37. Villaume K, Blanc M, Gouysse G, Walter T, Couderc C, Nejjari M, et al. VEGF secretion by neuroendocrine tumor cells is inhibited by octreotide and by inhibitors of the PI3K/AKT/mTOR pathway. Neuroendocrinology 2010;91:268-78.
38. Yazdani S, Kasajima A, Tamaki K, Nakamura Y, Fujishima F, Ohtsuka H, et al. Angiogenesis and vascular maturation in neuroendocrine tumors. Hum Pathol 2014;45:866-74.
39. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57.
40. Heinrich E, Trojan L, Friedrich D, Voss M, Weiss C, Michel MS, et al. Neuroendocrine tumor cells in prostate cancer:evaluation of the neurosecretory products serotonin, bombesin, and gastrindimpact on angiogenesis and clinical follow-up. Prostate 2011;71:1752-8.
41. Kim BJ, Zhou J, Martin B, Carlson OD, Maudsley S, Greig NH, et al. Transferrin fusion technology:a novel approach to prolonging biological half-life of insulinotropic peptides. J Pharmacol Exp Ther 2010;334:682-92.
42. Arnau J, Lauritzen C, Petersen GE, Pedersen J. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 2006;48:1-13.
43. Hu J, Cheng T, Zhang L, Sun B, Deng L, Xu H. Anti-tumor peptide AP25 decreases cyclin D1 expression and inhibits MGC-803 proliferation via phospho-extracellular signal-regulated kinase-, Src-, c-Jun N-terminal kinase- and phosphoinositide 3-kinase-associated pathways. Mol Med Rep 2015;12:4396-402.
44. Kim NA, An IB, Lim HS, Yang SI, Jeong SH. Biophysical evaluation of hybrid Fc fusion protein of hGH to achieve basal buffer system. Int J Pharm 2016;513:421-30.
45. Roopenian DC, Akilesh S. FcRn:the neonatal Fc receptor comes of age. Nat Rev Immunol 2007;7:715-25.
46. Bai Y, Shen WC. Improving the oral efficacy of recombinant granulocyte colony-stimulating factor and transferrin fusion protein by spacer optimization. Pharm Res 2006;23:2116-21.
47. Kellner C, Derer S, Klausz K, Rosskopf S, Wirt T, Rosner T, et al. Fc Glyco- and Fc protein-engineering:design of antibody variants with improved ADCC and CDC activity. Methods Mol Biol 2018;1827:381-97.
48. Huang Z, Zhang C, Chen S, Ye F, Xing XH. Active inclusion bodies of acid phosphatase PhoC:aggregation induced by GFP fusion and activities modulated by linker flexibility. Microb Cell Fact 2013;12:25.
49. Pemberton JG, Stafford JL, Chang JP. Ligand-selective signal transduction by two endogenous GnRH isoforms involves biased activation of the class I PI3K catalytic subunits p110β, p110γ, and p110δ in pituitary gonadotropes and somatotropes. Endocrinology 2015;156:218-30.
50. Aifa S, Frikha F, Miled N, Johansen K, Lundstrom I, Svensson SP. Phosphorylation of Thr654 but not Thr669 within the juxtamembrane domain of the EGF receptor inhibits calmodulin binding. Biochem Biophys Res Commun 2006;347:381-7.
51. Moretti RM, Monagnani Marelli M, van Groeninghen JC, Motta M, Limonta P. Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression. Endocr Relat Cancer 2003; 10:161-7.
52. Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A. Concise review:evidence for CD34 as a common marker for diverse progenitors. Stem Cells 2014;32:1380-9.