title: Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer.
abstract: BACKGROUND
  The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-gamma-seminoprotein E(4)B(7) murine mAb by guided selection.
  
  
  METHODS
  Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E(4)B(7) mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified gamma-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for gamma-seminoprotein were selected and further identified.
  
  
  RESULTS
  First, using the E(4)B(7) Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against gamma-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E(4)B(7) mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-gamma-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue.
  
  
  CONCLUSIONS
  Through guided-selection, we successfully produced a human anti-gamma-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.
authors.affiliation: Department of Clinical Laboratory, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, People's Republic of China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Si-He
authors.initials: SH
authors.lastname: Zhang
authors.forename: Ming-Quan
authors.initials: MQ
authors.lastname: Su
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Bao
authors.forename: Jia-Yun
authors.initials: JY
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Yi
authors.forename: Jian-Jun
authors.initials: JJ
authors.lastname: Shen
authors.forename: Xiao-Ke
authors.initials: XK
authors.lastname: Hao
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics*; D007150:Immunohistochemistry; D008297:Male; D008969:Molecular Sequence Data; D011355:Prodrugs / Q000138:chemical synthesis* / Q000494:pharmacology*; D017430:Prostate-Specific Antigen / Q000276:immunology*; D011471:Prostatic Neoplasms / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D007140:Immunoglobulin Fab Fragments; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D017430:Prostate-Specific Antigen