Shortened Hinge Design of Fab x sdAb-Fc Bispecific Antibodies Enhances Redirected T-Cell Killing of Tumor Cells

Biomolecules. 2022 Sep 21;12(10):1331. doi: 10.3390/biom12101331.

Abstract

T cell engager (TCE) antibodies have emerged as promising cancer therapeutics that link cytotoxic T-cells to tumor cells by simultaneously binding to CD3E on T-cells and to a tumor-associated antigen (TAA) expressed by tumor cells. We previously reported a novel bispecific format, the IgG-like Fab x sdAb-Fc (also known as half-IG_VH-h-CH2-CH3), combining a conventional antigen-binding fragment (Fab) with a single domain antibody (sdAb). Here, we evaluated this Fab x sdAb-Fc format as a T-cell redirecting bispecific antibody (TbsAbs) by targeting mEGFR on tumor cells and mCD3E on T cells. We focused our attention specifically on the hinge design of the sdAb arm of the bispecific antibody. Our data show that a TbsAb with a shorter hinge of 23 amino acids (TbsAb.short) showed a significantly better T cell redirected tumor cell elimination than the TbsAb with a longer, classical antibody hinge of 39 amino acids (TbsAb.long). Moreover, the TbsAb.short form mediated better T cell-tumor cell aggregation and increased CD69 and CD25 expression levels on T cells more than the TbsAb.long form. Taken together, our results indicate that already minor changes in the hinge design of TbsAbs can have significant impact on the anti-tumor activity of TbsAbs and may provide a new means to improve their potency.

Keywords: bispecific antibody; cancer immunotherapy; hinge; mCD3E; mEGFR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids
  • Antibodies, Bispecific* / chemistry
  • Cell Death
  • Humans
  • Immunoglobulin G
  • Neoplasms* / therapy
  • Single-Domain Antibodies*

Substances

  • Antibodies, Bispecific
  • Single-Domain Antibodies
  • Immunoglobulin G
  • Amino Acids

Grants and funding

This research was funded by the European Union, Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie Innovative Training Networks [grant number 765394, 2018]. Further support came from the Edinburgh Protein Production Facility (EPPF) and the Centre Core Grants (092076 and 203149) to the Wellcome Centre for Cell Biology at the University of Edinburgh.