Antibodies are a fast-growing area of interest in biology and medicine, and as such, the use of multivalent antibodies capable of binding multiple copies of an antigen is being explored for its positive effect on binding avidity and increased signaling efficacy.
Antibody nanocage (AbC) design/Image Credit: https://www.biorxiv.org/content/10.1101/2020.12.01.406611v1.full.pdf
A new study published in the bioRxiv* pre-print server shows the benefits of using computational design to create antibody nanocages that have the required orientation as well as a controlled number of binding sites, or valency. This could enable researchers to unite form with function in the creation of these designed antibodies.
With the market for antibodies, whether therapeutic or diagnostic, reaching $150 billion in 2019, there is an understandable eagerness to develop this field to its full potential. The advantage of using multivalent antibodies is the increased avidity of binding and the greater agonistic effects of receptor clustering.
Generating Multivalent Antibodies
At present, this is achieved by linking multiple antigen-binding fragments (Fab), IgM-derived fragment crystallizable (Fc) domain hexameric particles, IgM pentamers, fusing protein oligomers or nanoparticles to Ig-binding domains, or by linking several IgG dimers to inorganic materials.
In all of these, the functional domains are fused onto nanoparticle assemblies made of separate structural components. These attempts to create multimers are effective but also require extensive engineering of components or a multi-step conjugation process before the antibody oligomer is created.
Again, if nanoparticles are used, the flexible link to the Ig-binding domains makes it hard to ensure that all the binding sites are occupied and to keep the flocculation of the particles from happening after the binding of IgG dimers to multiple nanoparticles.
Meeting the Challenge: a Design Protein
Seeing the need for a technology that would ensure the ability to create multivalent antibody-based nanoparticles, conforming to a desired geometry and composition, the researchers attempted to design a protein that would result in an antibody-binding nanocage with twofold symmetry. Antibodies thus perform both structural and functional roles. This will bring multivalent antibodies on par with most commercially available IgG antibodies.
The first challenge is inducing the antibodies to assemble into symmetric nanobodies with a clear structure. They did this by using IgG molecules, with their two-fold symmetry, putting the symmetry axes on the two-fold axes of the final nanobody. They also designed another protein that would pin the IgGs in place.
This protein was created computationally from three types of monomers, Fc-binding proteins, helical linkers, and cyclic oligomers fused in a rigid structure. Each is important in the role it plays in the final fusion protein. The cyclic oligomer forms the basis of the cage-forming designer protein, linking the protein chains to form a nanocage interface. It ends in the Fc-binding protein that binds to the antibodies themselves, forming a second nanocage interface.
The linker joins both interfaces in the precise orientation needed to form antibody nanocages. The two-fold antibody axis and the oligomer symmetry axis are made to intersect at defined angles at the center of the structure to form the kind of architecture required. For instance, the angle would be 90 degrees and 31.7 degrees for dihedral and icosahedral nanocages, respectively.
The essential step to get right was having a large range of building blocks with enough potential fusion sites on each block to allow the very accurate geometrical symmetry of the nanocage to be attained. They used two Fc-binding proteins, 42 newly created helical monomers, and 1-3 oligomers, the number varying with the symmetry desired. With about 150 residues being available to fuse for each protein building block, there were about 107 potential fusions.
The shape of each of the three building blocks of the fusion protein, and the location as well as the geometry of the linking junctions, decide the rigid transform joining the two interfaces. All possible fusion builds were visualized computationally and the appropriate rigid body transforms for creating antibody nanocages with dihedral, tetrahedral, octahedral, and icosahedral architectures were thus identified.
Whatever the final shape, the dimeric antibody aligns across the two-fold axis of symmetry, but the designed oligomer protein ranges from a homodimer to a homopentamer. After further refining the shortlisted fusion models based on their closeness to the ideal build, they were tested for their structural rigidity and steric hindrance between backbone atoms. Following an optimization step to ensure rigid fusion geometry, they characterized 48 designs.
Rapid Formation of Antibody Assemblies
They found that 8 of these design proteins readily formed self-assemblies when mixed either with Fc or with full IgGs, though with the latter their flexibility was greater. At least 90% of the protein was taken up in the assembly. Structural analysis showed good agreement with the computational designed models. These 8 included dihedral, tetrahedral, octahedral, and icosahedral architectures, where the number of antibodies per nanocage was 2, 6, 12, and 30.
Both the cyclic oligomer and the nanocage could be designed simultaneously, though with somewhat smaller success rates, generating antibody nanocages with D3 symmetry. The researchers also found that their nanocages were stable for 3-5 weeks at room temperatures, except for one, and did not show any evidence of the exchange of the Fc-containing component with another Fc or IgG, as could be the case if such molecules were used to treat patients with high serum IgG levels.
Enhanced Activation of Receptor-Mediated Signaling Pathways
Using this wide set of designed antibody nanocages, the investigators explored the effect of multivalency and the geometric symmetry of receptor binding on the activation of signaling pathways. They generated nanocages from antibodies and Fc-fusion proteins affecting multiple signaling pathways, to find their effects on signaling, using the widest possible cage symmetries in each pathway test.
They found that these design proteins activated many signaling pathways, such as d tumor cell apoptosis when bivalent DR5 is linked to trimeric TNF-related apoptosis-inducing ligand (TRAIL). In contrast, the bare antibody, or nanocages formed of Fc alone, failed to induce cell death at the highest concentrations. Resistance was not found to emerge after 6 days of treatment, and apoptosis did not occur in healthy cell lines.
Similarly, the antibody nanocages generated against specific cell surface receptors improved the intensity of signaling relative to the effect of either free antibodies or Fc-containing fusion proteins. This was observed in Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation.
Tie2 is an angiopoietin-1 receptor and its activation could be useful in improved wound healing. The TNF- receptor superfamily member CD40 is found on dendritic and B cells, and when cross-linked by its trimeric ligand on T cells activates the signaling pathway ending in cell proliferation. Non-agonistic ⍺-CD40 antibodies when formed into nanocages were able to induce strong CD40 activation without the need to be present on the cell surface.
Increased Neutralization Capacity
A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization test using ⍺-SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins showed increased neutralization capacity, as expected since the multivalent presentation of the anti-SARS-CoV-2 antibodies into nanocages is expected to increase the avidity of antibodies for the viral particles. When Fc-ACE2 fusion protein was formed into octahedral assemblies, the neutralization capacity for SARS-CoV-2 was enhanced sevenfold compared to free Fc-ACE2.
What are the Implications?
We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have a broad impact in biology and medicine.”
Using this strategy, other homo-oligomers could be used as long as they have the right terminal components, such as helical antigen-binding proteins to fuse viral glycoproteins, or symmetric enzymes with free helices to boost the location of active sites involved in consecutive reactions.
The modular approach allows practically any known antibody to be picked up and assembled with the right design protein that offers adequate affinity of binding between the protein and the Fc binding component. The degree of control it offers concerning the positioning and valency of the Fc binding domain is unsurpassed by other antibody-protein nanoparticles.
Their applications are also diverse, offering signaling pathway agonism with a high degree of structural homogeneity, which results in more predictable phenotypic effects, and thus better control of the process.
Mixing of Fc-fusions or antibodies targeting different receptors can generate nanocages containing both to assess the effects of the mixture on downstream signaling pathways, at different valencies and architectures. They could also be used to deliver protein or nucleic acid cargo, raising the possibility of antibody therapeutics. This will require further studies into their pharmacological properties.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.