Effective targeting of prostate cancer by lymphocytes redirected by a PSMA × CD3 bispecific single-chain diabody

Prostate. 2011 May;71(6):588-96. doi: 10.1002/pros.21274. Epub 2010 Oct 13.


Background: For redirecting T-lymphocytes to induce prostate cancer cell lysis, we constructed a novel bispecific single-chain (bsc) diabody directed to the prostate specific membrane antigen (PSMA) and the T-cell receptor (TCR)-associated CD3 molecule on T-cells.

Methods: The PSMA × CD3 bsc diabody was generated from an anti-CD3 single chain Fv fragment (scFv) and the anti-PSMA scFv D7. It was expressed in E. coli and purified from the periplasmic extract and culture supernatant by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST-1) was used and activation of T-cells was determined by measuring the surface marker expression of CD25 and CD69. For in vivo evaluation, the diabody was administered in combination with human peripheral blood lymphocytes (Ly) in a C4-2 xenograft-SCID mouse model.

Results: Specific binding of the PSMA × CD3 bsc diabody both to CD3-positive Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the PSMA × CD3 bsc diabody proved to be a potent agent for retargeting CD4+ and CD8+ human lymphocytes to lyse C4-2 prostate cancer cells. Treatment of SCID mice bearing C4-2 tumor xenografts with the diabody and human lymphocytes efficiently inhibited tumor growth.

Conclusions: The PSMA × CD3 bsc diabody bears a high potential for the immunotherapy of prostate cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Bispecific / genetics
  • Antibodies, Bispecific / immunology*
  • Antigens, Surface / genetics
  • Antigens, Surface / immunology*
  • Biological Assay
  • Blotting, Western
  • CD3 Complex / genetics
  • CD3 Complex / immunology*
  • Cell Growth Processes / immunology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Flow Cytometry
  • Glutamate Carboxypeptidase II / genetics
  • Glutamate Carboxypeptidase II / immunology*
  • Humans
  • Immunotherapy, Adoptive / methods*
  • Jurkat Cells
  • Male
  • Mice
  • Mice, SCID
  • Prostatic Neoplasms / immunology
  • Prostatic Neoplasms / therapy*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / immunology
  • T-Lymphocytes, Cytotoxic / immunology*


  • Antibodies, Bispecific
  • Antigens, Surface
  • CD3 Complex
  • Recombinant Proteins
  • Single-Chain Antibodies
  • FOLH1 protein, human
  • Glutamate Carboxypeptidase II