Development and characterization of a bispecific single-chain antibody directed against T cells and ovarian carcinoma

Hybridoma. 2000 Feb;19(1):33-41. doi: 10.1089/027245700315770.


Bispecific antibodies with specificity for tumor antigen and CD3 have been shown to redirect the cytotoxicity of T cells against relevant tumor. Our objective was to generate single-chain bispecific antibodies (bsSCA) that could retarget mouse cytotoxic T lymphocytes (CTL) to destroy human ovarian carcinoma in a xenogeneic setting. A bsSCA, 2C11 x B43.13, was constructed by genetic engineering and expressed in mammalian cells. Molecular characteristics, binding properties, and ability to retarget CTL were studied. Western blot analysis showed that the product is a 65-kDa protein. Purification of antibodies could be done by single-step affinity chromatography using protein L-agarose with an unoptimized yield of 200 microg/L. BsSCA 2C11 x B43.13 was capable of binding to mouse CD3 and human CA125 as detected by FACS analysis of EL4 and OVCAR Nu3H2 cells, respectively. It could also bridge activated splenic T cells and human ovarian carcinoma as demonstrated by a bridge FACS assay. Redirected mouse CTL could mediate human target cell lysis in a 20-h 51Cr release assay despite that they are xenogeneic. Prolonged incubation of redirected CTL and tumor targets resulted in a dramatic reduction in tumor cell number. CD28 co-stimulation enhanced redirected CTL function in both types of assays. BsSCA 2C11 x B43.13 thus can be used as a preclinical immunotherapeutic model for human ovarian cancer in a xenogeneic setting.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Bispecific / biosynthesis
  • Antibodies, Bispecific / genetics
  • Antibodies, Bispecific / isolation & purification*
  • Antibodies, Bispecific / toxicity
  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal, Murine-Derived
  • CA-125 Antigen / immunology
  • CD3 Complex / immunology
  • Coculture Techniques
  • Cytotoxicity, Immunologic
  • Female
  • Growth Inhibitors / biosynthesis
  • Growth Inhibitors / genetics
  • Growth Inhibitors / isolation & purification
  • Growth Inhibitors / toxicity
  • Humans
  • Mice
  • Mice, Inbred BALB C
  • Mice, Nude
  • Ovarian Neoplasms / immunology*
  • Ovarian Neoplasms / pathology
  • Ovarian Neoplasms / therapy
  • T-Lymphocytes / immunology*
  • Tumor Cells, Cultured


  • Antibodies, Bispecific
  • Antibodies, Monoclonal
  • Antibodies, Monoclonal, Murine-Derived
  • CA-125 Antigen
  • CD3 Complex
  • Growth Inhibitors
  • oregovomab