title: rG17PE38, a novel immunotoxin target to gastric cancer with overexpressed CCK-2R.
abstract: BACKGROUND
  Gastrin/cholecystokinin subtype 2 receptor (CCK2R) is overexpressed in several types of tumors. Gastrin-17 (G17) peptide has a high affinity with CCK2R. These characters suggest that G17 may be useful for target cancer therapy.
  
  
  OBJECTIVE
  Construct a new immunotoxin (IT) targeting of CCK2R overexpressed gastric cancer.
  
  
  METHODS
  Two ITs were generated using forward and reverse G17 peptides fused with PE38. To get a high yield, codon optimized gene and optimized fermentation parameters were used in large-scale protein expression. An immunoaffinity technique was introduced into pseudomonas exotoxin (PE)-derived IT purification procedure. G17 competition, GST pull-down and indirect immunoflourescence assays were carried out to confirm the interaction between rG17 and CCK2R. Then, several cytotoxic assays were carried out on 18 cell lines, and an in vivo antitumor activity experiment was tested in nude mice.
  
  
  RESULTS
  The rG17PE38 showed specific cytotoxicity on three gastric cancer cells, while G17PE38 did not. After optimization, the expression level reached about 40% in medium deprived of NaCl. Next, 15-27.5 mg of pure rG17PE38 per 1 L of cultures was obtained. Results of G17 competition, GST pull-down and indirect immunoflourescence assays demonstrated that rG17 have a specific interact with CCK2R. Purified rG17PE38 showed high cytotoxicity on gastric cancer cell lines with the IC50 value of 0.6-4 ng·mL(-1). Treatment of nude mice inoculated with BGC-823 tumor xenografts with rG17PE38 efficiently inhibited tumor size.
  
  
  CONCLUSIONS
  The present study demonstrates that reversed G17 could be used as target moiety of PE-derived IT and the rG17PE38 could be developed as a new immunotherapy agent. Codon optimized gene could increase the rG17PE38 expression level in E. coli and furthermore NaCl inhibits the rG17PE38 expression in large scale. Meanwhile, our present study inducts an immunoaffinity method in the IT purification procedure, which could purify the PE-derived ITs in native form.
authors: Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, PR China.
authors: Jie
authors: J
authors: Song
authors: Honglin
authors: H
authors: Ren
authors: Yansong
authors: Y
authors: Li
authors: Junyan
authors: J
authors: Xu
authors: Hong
authors: H
authors: Kong
authors: Weihua
authors: W
authors: Tong
authors: Yu
authors: Y
authors: Zhou
authors: Shiqi
authors: S
authors: Gao
authors: Yanyan
authors: Y
authors: Liu
authors: Qi
authors: Q
authors: Hui
authors: Qisheng
authors: Q
authors: Peng
authors: Shiying
authors: S
authors: Lu
authors: Zengshan
authors: Z
authors: Liu
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003062:Codon; D005260:Female; D005755:Gastrins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D000922:Immunotoxins / Q000493:pharmacokinetics* / Q000494:pharmacology*; D020014:K562 Cells; D061986:MCF-7 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D043887:Receptor, Cholecystokinin B / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D013274:Stomach Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D003062:Codon; D005755:Gastrins; D000922:Immunotoxins; D043887:Receptor, Cholecystokinin B; D012965:Sodium Chloride; C013550:gastrin 17