Research Article

Designed proteins assemble antibodies into modular nanocages

See allHide authors and affiliations

Science  02 Apr 2021:
Vol. 372, Issue 6537, eabd9994
DOI: 10.1126/science.abd9994

You are currently viewing the abstract.

View Full Text

Log in to view the full text

Log in through your institution

Log in through your institution

Integrating form and function for design

Antibodies are broadly used in therapies and as research tools because they can be generated against a wide range of targets. Efficacy can often be increased by clustering antibodies in multivalent assemblies. Divine et al. designed antibody nanocages from two components: One is an antibody-binding homo-oligomic protein and the other is the antibody itself. Computationally designed proteins drive the assembly of antibody nanocages in a range of architectures, allowing control of the symmetry and the antibody valency. The multivalent display enhances antibody-dependent signaling, and nanocages displaying antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein effectively neutralize pseudovirus.

Science, this issue p. eabd9994

Structured Abstract

INTRODUCTION

Antibodies that bind tightly to targets of interest play central roles in biological research and medicine. Clusters of antibodies, typically generated by fusing antibodies to polymers or genetically linking antibody fragments together, can enhance signaling. Currently lacking are approaches for making antibody assemblies with a range of precisely specified architectures and valencies.

RATIONALE

We set out to computationally design proteins that assemble antibodies into precise architectures with different valencies and symmetries. We developed an approach to designing proteins that position antibodies or Fc-fusions on the twofold symmetry axes of regular dihedral and polyhedral architectures. We hypothesized that such designs could robustly drive arbitrary antibodies into homogeneous and structurally well-defined nanocages and that such assemblies could have pronounced effects on cell signaling.

RESULTS

Antibody cage (AbC)–forming designs were created by rigidly fusing antibody constant domain–binding modules to cyclic oligomers through helical spacer domains such that the symmetry axes of the dimeric antibody and cyclic oligomer are at orientations that generate different dihedral or polyhedral (e.g., tetrahedral, octahedral, or icosahedral) architectures. The junction regions between the connected building blocks were optimized to fold to the designed structures. Synthetic genes encoding the designs were expressed in bacterial cultures; of 48 structurally characterized designs, eight assemblies matched the design models. Successful designs encompass D2 dihedral (three designs), T32 tetrahedral (two designs), O42 octahedral (one design), and I52 icosahedral (two designs) architectures; these contain 2, 6, 12, or 30 antibodies, respectively.

We investigated the effects of AbCs on cell signaling. AbCs formed with a death receptor–targeting antibody induced apoptosis of tumor cell lines that were unaffected by the soluble antibody or the native ligand. Angiopoietin pathway signaling, CD40 signaling, and T cell proliferation were all enhanced by assembling Fc-fusions or antibodies in AbCs. AbC formation also enhanced in vitro viral neutralization of a severe acute respiratory syndrome coronavirus 2 pseudovirus.

CONCLUSION

We have designed multiple antibody cage–forming proteins that precisely cluster any protein A–binding antibody into nanocages with controlled valency and geometry. AbCs can be formed with 2, 6, 12, or 30 antibodies simply by mixing the antibody with the corresponding designed protein, without the need for any covalent modification of the antibody. Incorporating receptor binding or virus-neutralizing antibodies into AbCs enhanced their biological activity across a range of cell systems. We expect that our rapid and robust approach for assembling antibodies into homogeneous and ordered nanocages without the need for covalent modification will have broad utility in research and medicine.

Designed proteins assemble antibodies into large symmetric architectures.

Designed antibody-clustering proteins (light gray) assemble antibodies (purple) into diverse nanocage architectures (top). Antibody nanocages enhance cell signaling compared with free antibodies (bottom).

IMAGE: IAN HAYDON, INSTITUTE FOR PROTEIN DESIGN

Abstract

Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)–mediated apoptosis, angiopoietin-1 receptor (Tie2)–mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc–angiotensin-converting enzyme 2 (ACE2) fusion proteins.

View Full Text

Stay Connected to Science