title: Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-β subunit.
abstract: OBJECTIVE
  Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHβ-Fc/Fc, offer a potential FSH alternative?
  
  
  CONCLUSIONS
  KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy.
  
  
  BACKGROUND
  rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed.
  
  
  METHODS
  Plasmids containing FSHα, FSHβ-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH.
  
  
  METHODS
  The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation.
  
  
  RESULTS
  KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSH's activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH.
  
  
  CONCLUSIONS
  All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans.
  
  
  CONCLUSIONS
  KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications.
  
  
  BACKGROUND
  This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.
authors.affiliation: Life Sciences Institute, Zhejiang University, Hangzhou 310058, China.
authors.forename: Yin-Li
authors.initials: YL
authors.lastname: Zhang
authors.affiliation: Alphamab Co. Ltd., Suzhou 215125, China.
authors.forename: Kang-Ping
authors.initials: KP
authors.lastname: Guo
authors.affiliation: Life Sciences Institute, Zhejiang University, Hangzhou 310058, China.
authors.forename: Shu-Yan
authors.initials: SY
authors.lastname: Ji
authors.affiliation: Life Sciences Institute, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xiao-Man
authors.initials: XM
authors.lastname: Liu
authors.affiliation: Alphamab Co. Ltd., Suzhou 215125, China.
authors.forename: Pilin
authors.initials: P
authors.lastname: Wang
authors.affiliation: Alphamab Co. Ltd., Suzhou 215125, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.affiliation: Alphamab Co. Ltd., Suzhou 215125, China.
authors.forename: Li
authors.initials: L
authors.lastname: Gao
authors.affiliation: Alphamab Co. Ltd., Suzhou 215125, China.
authors.forename: Tian-Qi
authors.initials: TQ
authors.lastname: Jiang
authors.affiliation: Alphamab Co. Ltd., Suzhou 215125, China hyfan@zju.edu.cn tingxu@alphamab.com.
authors.forename: Ting
authors.initials: T
authors.lastname: Xu
authors.affiliation: Life Sciences Institute, Zhejiang University, Hangzhou 310058, China hyfan@zju.edu.cn tingxu@alphamab.com.
authors.forename: Heng-Yu
authors.initials: HY
authors.lastname: Fan
mesh_terms: D000818:Animals; D006224:Cricetinae; D005260:Female; D005640:Follicle Stimulating Hormone / Q000819:agonists*; D037201:Follicle Stimulating Hormone, beta Subunit; D008252:Macaca fascicularis; D051379:Mice; D008813:Mice, Inbred ICR; D010053:Ovary / Q000187:drug effects*; D010060:Ovulation / Q000187:drug effects*; D010446:Peptide Fragments; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics* / Q000494:pharmacology*
chemical_list: D037201:Follicle Stimulating Hormone, beta Subunit; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005640:Follicle Stimulating Hormone
keywords: follicle growth; follicle stimulating hormone; granulosa cell; oocyte; ovulation