Solid-Phase Bioconjugation of Heterobifunctional Adaptors for Versatile Assembly of Bispecific Targeting Ligands

Abstract

High-throughput generation of bispecific molecules promises to expedite the discovery of new molecular therapeutics and guide engineering of novel multifunctional constructs. However, high synthesis complexity and cost have hampered the discovery of bispecific molecules in drug development and biomedical research. Herein we describe a simple solid-phase bioconjugation procedure for preparation of Protein A(G,L)-PEG-Streptavidin heterobifunctional adaptors (with 1:1:1 stoichiometry), which enable self-assembly of unmodified antibodies and biotinylated molecules into bispecific targeting ligands in a versatile mix-and-use manner. Utility of such adaptors is demonstrated by assembly of anti-CD3 and anti-Her2 antibodies into bispecific CD3xHer2 targeting ligands, which efficiently drive T-cell-mediated lysis of Her2-positive cancer cells. In comparison to bioconjugation in solution, the solid-phase procedure described here offers precise stoichiometry control, ease of purification, and high yield of functional conjugates. Simplicity and versatility should prove this methodology instrumental for preparation of bispecific ligands, as well as for high-throughput screening of bispecific combinations, before proceeding to synthesis of lead candidates via recombinant engineering or chemical cross-linking.

Functional protein nanostructures: a chemical toolbox

“…Both Abs, anti-Her2 and anti-CD3, were assembled on the Protein A–PEG–streptavidin adaptor described in Section 2.3.3. to form the bispecific Ab. Flow cytometry assays confirmed that the Ab bispecific construct was able to target both Her2-positive human breast cancer SKBR3 cells in combination with CD3-positive human peripheral blood mononuclear cells, which served as the effector cells 199. Therefore, the CD3xHer2 bispecific Ab had an effect on T cell-mediated lysis of Her2-positive breast cancer cells.…”

“…Thus, it is intuitive that the formation of trimeric or oligomeric heteroproteins would require much more synthetic efforts. Nevertheless, there has been some recent breakthrough with the development of solid phase preparation of protein nanoarchitectures 19,20,199. Solid phase protein synthesis approaches have been devised to desymmetrize homomeric protein building blocks such as (strept)avidin 19,20.…”

“…11a). 199 First, monomeric avidin resin was loaded with biotin–PEG–amine, followed by addition of NHS-activated Protein A to form monovalent Protein A–PEG–biotin conjugates. After elution of the Protein A–PEG–biotin conjugate from the column, streptavidin was added to Protein A–PEG–biotin immobilized onto a human immunoglobulin G agarose column.…”

“…Protein A can also be replaced by Protein L or G to expand the library of the available trimeric adaptor. This adaptor was characterized using gel electrophoresis, and it was further functionalized on its two ends with bioactive components 199. Notably, the authors prepared a hetero-pentameric complex where two antibodies were further conjugated to the Protein A–PEG–streptavidin adaptor.…”

“…Notably, the authors prepared a hetero-pentameric complex where two antibodies were further conjugated to the Protein A–PEG–streptavidin adaptor. The successful conjugation was characterized by the preservation of both antibodies’ functions through the antibody-mediated uptake into on Her2-positive human breast cancer cells (SKBR3) and CD3-positive human peripheral blood mononuclear cells 199…”