Nanocell targeting using engineered bispecific antibodies |
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Authors: | Karin Taylor Christopher B Howard Martina L Jones Ilya Sedliarou Jennifer MacDiarmid Himanshu Brahmbhatt Trent P Munro Stephen M Mahler |
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Affiliation: | 1.Australian Institute for Bioengineering and Nanotechnology (AIBN); University of Queensland, St Lucia; Queensland, Australia;2.EnGeneIC Ltd; New South Wales, Australia;3.School of Chemical Engineering; University of Queensland, St Lucia; Queensland, Australia |
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Abstract: | There are many design formats for bispecific antibodies (BsAbs), and the best design choice is highly dependent on the final application. Our aim was to engineer BsAbs to target a novel nanocell (EnGeneIC Delivery Vehicle or EDVTMnanocell) to the epidermal growth factor receptor (EGFR). EDVTMnanocells are coated with lipopolysaccharide (LPS), and BsAb designs incorporated single chain Fv (scFv) fragments derived from an anti-LPS antibody (1H10) and an anti-EGFR antibody, ABX-EGF. We engineered various BsAb formats with monovalent or bivalent binding arms and linked scFv fragments via either glycine-serine (G4S) or Fc-linkers. Binding analyses utilizing ELISA, surface plasmon resonance, bio-layer interferometry, flow cytometry and fluorescence microscopy showed that binding to LPS and to either soluble recombinant EGFR or MDA-MB-468 cells expressing EGFR, was conserved for all construct designs. However, the Fc-linked BsAbs led to nanocell clumping upon binding to EDVTMnanocells. Clumping was eliminated when additional disulfide bonds were incorporated into the scFv components of the BsAbs, but this resulted in lower BsAb expression. The G4S-linked tandem scFv BsAb format was the optimal design with respect to EDV binding and expression yield. Doxorubicin-loaded EDVTMnanocells actively targeted with tandem scFv BsAb in vivo to MDA-MB-468-derived tumors in mouse xenograft models enhanced tumor regression by 40% compared to passively targeted EDVTMnanocells. BsAbs therefore provide a functional means to deliver EDVTMnanocells to target cells. |
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Keywords: | bispecific antibody disulfide bridge mammalian expression nanoparticle single chain Fv surface plasmon resonance tumor regression |
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