Construction, expression and binding specificity of bispecific CD3 × VEGFR-2 and CD3 × NCAM antibodies in the single chain and diabody format — Oak Academic Publishing
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Construction, expression and binding specificity of bispecific CD3 × VEGFR-2 and CD3 × NCAM antibodies in the single chain and diabody format
Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
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Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
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Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
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Department of Pediatric Oncology and Hematology, University of Cologne, University Hospital Cologne, Cologne, Germany
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Department of Pediatric Oncology and Hematology, University of Cologne, University Hospital Cologne, Cologne, Germany
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Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
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Institute for Cell Biology, Department of Immunology, Eberhard Karls University, Tübingen, Germany
1 Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
2 Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
3 Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
4 Department of Pediatric Oncology and Hematology, University of Cologne, University Hospital Cologne, Cologne, Germany
5 Department of Pediatric Oncology and Hematology, University of Cologne, University Hospital Cologne, Cologne, Germany
6 Helmholtz Center for Infection Research, Department of Molecular Biotechnology, Braunschweig, Germany
7 Institute for Cell Biology, Department of Immunology, Eberhard Karls University, Tübingen, Germany
Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different va riable domain combinations with respect to the paren tal monospecific single chain fragments (scFv) were evaluated after expression in bacteria or mammalian cells. The parental scFv proteins humanized anti- NCAM scFv, murine anti-VEGFR-2 scFv, murine and humanized anti-CD3 scFv , respectively, could success fully be expressed in E. coli , whereas the murine anti-NCAM scFv version could not be reliably detected. Bispecific CD3 × VEGFR-2 and CD3 × NCAM anti-bodies were expressed in the bispecific single chain and the single chain diabody format. However, the diabody derived from the murine anti-NCAM scFv could not efficiently be expressed in E. coli or in mam malian cells. Significant binding of the CD3 × NCAM single chain diabody comprising the humanized version of anti-CD3 and humanized version of anti-NCAM was efficient to both antigens. Nevertheless, binding of the bispecific single chain version to the NCAM antigen was inefficient in comparison to CD3 binding. In conclusion, the data could indicate that the result of scFv expression in bacteria may be predictive for the chances of success for functional expression of more complex bispecific derivatives.
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