title: RP215 single chain fragment variable and single domain recombinant antibodies induce cell cycle arrest at G0/G1 phase in breast cancer.
abstract: BACKGROUND
  Targeted therapy is an attractive approach to avoid the side effects of cancer treatment. Based on antibody-targeted superantigens, single chain variable fragment (scFv) and single domain (sdAb) antibodies, characterized by a low molecular weight, low immunogenicity and a high tumor penetration compared to monoclonal antibodies (mAb), have been increasingly used in gene-targeted therapy for cancer. In the present study, we aimed to develop the novel recombinant scFv-RP215 and sdAb-RP215 antibodies based on the variable regions of the RP215 monoclonal antibody (RP215-mAb) against CA215, a pan cancer marker expressed in various human tumor tissues, and to examine their biological activity in breast cancer cell lines.
  
  
  METHODS
  The VH and VL genes were amplified from hybridoma cells secreting RP215-mAb by RT-PCR and joined with a linker using splicing by overlap extension PCR (SOE-PCR) to obtain the RP215-scFv gene, whereas the VH gene was used to generate the RP215-sdAb. Gene fragments of antibodies were subcloned into the pET32a(+) vector and expressed in Escherichia coli BL21. Western blot, indirect immunofluorescence (IF), ELISA and competitive ELISA were used to detect the immunoreactivity of scFv-RP215, sdAb-RP215, and RP215-mAb. The CCK-8 assay and cell cycle analysis were used to assess antibodies function.
  
  
  RESULTS
  The novel recombinant scFv-RP215 and sdAb-RP215 antibodies were successfully developed based on the variable regions of the monoclonal antibody RP215 (RP215-mAb) against CA215. We verified that scFv-RP215 and sdAb-RP215 recognize CA215 on the surface of breast cancer cells (MB231, MCF7, MB468, SK-BR-3 and BT549) and characterized their activity and specificity. Our findings also indicate that scFv-RP215 and sdAb-RP215 induce cell cycle arrest at the G0/G1 phase in breast cancer cells.
  
  
  CONCLUSIONS
  Our results showed that scFv-RP215 and sdAb-RP215 have excellent immunoreactivity and localize accurately to breast cancer cells in membrane-bound form, suggesting their potential as tumor targeting antibodies for breast cancer therapy.
authors.affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Fang
authors.initials: F
authors.lastname: Yu
authors.affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China; Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xiao
authors.affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Orthopaedics, The Children's Hospital of Chongqing Medical University, 400016, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Luo
authors.affiliation: Department of Nuclear Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Dong
authors.initials: D
authors.lastname: Duan
authors.affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Chunhong
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Radiology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
authors.forename: Shengsheng
authors.initials: S
authors.lastname: Xu
authors.affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China. Electronic address: xiangtx1@gmail.com.
authors.forename: Tingxiu
authors.initials: T
authors.lastname: Xiang
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D000951:Antigens, Neoplasm / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D059447:Cell Cycle Checkpoints / Q000276:immunology*; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D016193:G1 Phase / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D016192:Resting Phase, Cell Cycle / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; C541379:CA215 antigen, human; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D061905:Single-Domain Antibodies
keywords: Breast cancer; Targeted therapy; scFv-RP215; sdAb-RP215