“BIClonals”: Production of Bispecific Antibodies in IgG Format in Transiently Transfected Mammalian Cells

  • Dana Litvak-Greenfeld
  • Lilach Vaks
  • Stav Dror
  • Limor Nahary
  • Itai BenharEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1904)


Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components. For some therapeutic applications, full-size, native IgG-like bsAbs may be the optimal format.

To prepare bsAbs in IgG format, two challenges should be met. One is that each heavy chain will only pair with the heavy chain of the second specificity and that heavy chain homodimerization will be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and that pairing with the light chain of the second specificity will be prevented. The first solution to the first criterion (known as knobs into holes, KIH) was presented in 1996 by Genentech and additional solutions were presented more recently. However, until recently, out of >120 published formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested.

Here, we present a protocol for preparing bsAbs in IgG format in transfected mammalian cells. For heavy chain dimerization we use KIH while as a solution for the second challenge—correct pairing of heavy and light chains of bispecific IgGs we present our “BIClonals” technology; an engineered (artificial) disulfide bond between the antibodies’ variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL.

During our studies of bsAbs we found that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies; hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs that suits every antibody sequence, making it necessary to carefully evaluate the optimal solution for each new antibody.

Key words

CDR Complementarity-determining region dsFv Disulfide-stabilized Fv fragment An IgG heavy chain KIH Knobs-into-holes An IgG light chain mAb Monoclonal antibody VH Variable domain of the heavy chain VL Variable domain of the light chain 



Studies of bispecific antibodies at the Authors’ lab were supported in part by The Israel Science Foundation (Grant no. 591/13), by a research grant from the Israel Cancer Research fund (ICRF), by a grant from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics, by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund and by Varda and Boaz Dotan Research Center in Hemato-oncology affiliated to CBRC at Tel-Aviv University. We are grateful to members of the Benhar Lab for their contributions in optimizing the BIClonals technology.


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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Dana Litvak-Greenfeld
    • 1
  • Lilach Vaks
    • 1
  • Stav Dror
    • 1
  • Limor Nahary
    • 1
  • Itai Benhar
    • 1
    Email author
  1. 1.School of Molecular Cell Biology and Biotechnology, The George S. Wise Faculty of Life SciencesTel-Aviv UniversityRamat AvivIsrael

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