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- Title
Guided selection of an anti-gamma-seminoprotein human Fab for antibody directed enzyme prodrug therapy of prostate cancer.
- Authors
Qing Zhang; Si-He Zhang; Ming-Quan Su; Guo-Qiang Bao; Jia-Yun Liu; Jing Yi; Jian-Jun Shen; Xiao-Ke Hao
- Abstract
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-γ-seminoprotein E4B7 murine mAb by guided selection. 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 E4B7 mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified γ-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 γ-seminoprotein were selected and further identified. First, using the E4B7 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 γ-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 E4B7 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-γ-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue. Through guided-selection, we successfully produced a human anti-γ-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.
- Subjects
PROSTATE cancer; SEMINAL proteins; CANCER cells; IMMUNOGLOBULINS; ANTIGENS; IMMUNOHISTOCHEMISTRY; FLOW cytometry
- Publication
Cancer Immunology, Immunotherapy, 2007, Vol 56, Issue 4, p477
- ISSN
0340-7004
- Publication type
Article
- DOI
10.1007/s00262-006-0202-2