Nanocell targeting using engineered bispecific antibodies

• Published in: mAbs Vol. 7; no. 1; pp. 53 - 65
• Main Authors: Taylor, Karin, Howard, Christopher B, Jones, Martina L, Sedliarou, Ilya, MacDiarmid, Jennifer, Brahmbhatt, Himanshu, Munro, Trent P, Mahler, Stephen M
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 01.01.2015
• Subjects: Animals; Antibodies, Bispecific - chemistry; Antibodies, Bispecific - genetics; Antibodies, Bispecific - immunology; Antibodies, Bispecific - pharmacology; Antibodies, Neoplasm - chemistry; Antibodies, Neoplasm - genetics; Antibodies, Neoplasm - immunology; Antibodies, Neoplasm - pharmacology; Breast Neoplasms - drug therapy; Breast Neoplasms - immunology; CHO Cells; Cricetinae; Cricetulus; Drug Delivery Systems; Female; Humans; Mice; Mice, Nude; Receptor, Epidermal Growth Factor - immunology; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - pharmacology; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - immunology; Single-Chain Antibodies - pharmacology; Xenograft Model Antitumor Assays; mammalian expression; NP, nanoparticle; mAb, monoclonal antibody; nanoparticle; single chain Fv; BsAb, bispecific antibody; EGFR, epidermal growth factor receptor; IgG, immunoglobulin G; surface plasmon resonance; scFv, single chain variable fragment; bispecific antibody; LPS, lipopolysaccharide; disulfide bridge; tumor regression; EDVTM, EnGeneIC Delivery Vehicle
• DOI: 10.4161/19420862.2014.985952
• ISSN: 1942-0862

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 EDV(TM)nanocell) to the epidermal growth factor receptor (EGFR). EDV(TM)nanocells 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 EDV(TM)nanocells. 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 EDV(TM)nanocells 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 EDV(TM)nanocells. BsAbs therefore provide a functional means to deliver EDV(TM)nanocells to target cells.