From monoclonals to bi-specifics: Harnessing the transformative power of VHHs to supercharge IgG therapeutics
Updated on Feb. 16th, 2025 – Since their discovery over thirty years ago, monoclonal antibodies (mAbs) based on Immunoglobulin G (IgG) have transformed the diagnosis and treatment of many diseases.
In particular, IgGs targeting the programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway have made the promises of cancer immunotherapy a reality.
However, despite there now being over 100 FDA-approved mAbs on the market, these well-characterised drugs have yet to live up to their full potential.
The quest for better biologics
IgG-based immunotherapies and targeted therapies have made a profound impact on the survival rates for some cancers over the past couple of decades.
However, many cancers do not respond effectively to these treatments. This is due to a range of factors, from the limited number of well-characterised tumour-specific antigens to the impact of alternating suppressive pathways on cancer cell survival.
At the heart of these issues lies the inherent nature of monospecific, monoclonal antibodies. An antibody that targets just one protein makes it much easier for a tumour to evolve resistance to therapy. If a small number of mutated cancer cells lose expression of the protein target, these cells are no longer responsive to the treatment and can proliferate unchecked.
With these significant drawbacks in mind, researchers have increasingly turned to an innovative solution in the form of bi- or multi-specific antibodies, which target two or more proteins.
The beauty of bi-specificity
Bi-specific antibodies (bsAb) or multi-specific antibodies are designed to bind to two or more antigens, opening the door to a whole new world of therapeutic potential.
For example, in a 2020 study by Kotanides et al., researchers at Eli Lilly investigated the possibility of co-targeting PD-1 and PD-L1 with a bi-specific IgG1 antibody, LY3434172.
They found that LY3434172 exhibited strong, dose-dependent anti-tumour activity and enhanced T cell activation compared to mono- or combination therapy. Overall, the drug showed enhanced immunomodulatory properties and better anti-tumour activity compared with monospecific approaches. Recently, LY3434172 has shown preliminary efficacy in phase I clinical trials.
VHHs: Fantastic flexibility for IgG enhancement
VHHs – small format single-domain antibody fragments – have a range of unique properties that make them ideal for enhancing the therapeutic potential of conventional IgG antibodies.
A major challenge in traditional bispecific IgG antibody development is the risk of chain mispairing, which necessitates extensive protein engineering strategies such as knobs-into-holes (KiH), charge-pair-mutations (CPMs), or orthogonal mutations to enforce correct assembly.
One alternative is adding a VHH domain to an IgG antibody, which can expand the antigen binding repertoire of an IgG by increasing valency toward a specific target or enabling the creation of bi-, tri-or tetra-specific antibodies — all while retaining its Fc effector function and the benefits of prolonged half-life from FcRn recycling.
VHHs are also more stable and soluble compared to other antibody fragments such as scFvs. This creates a simpler or “modular” molecule that can easily be bolted to the terminals of the heavy or light chains of an antibody, while preserving the symmetry of the IgG structure, thereby significantly reducing the risk of chain mispairing.
Where IgG-VHH fusions like this have been created (view reference), the molecules have retained high stability, and comparable or higher yields compared to IgG alone.
Isogenica Case Study
With a decade of synthetic VHH discovery campaigns under our belt, it’s only natural we’d want to see how these single-domain antibodies can be used to upskill an IgG. Using a similar approach to the Eli Lilly team, we constructed a bi-specific PD-1 x PD-L1 antibody by fusing the blockbuster anti-PD-1 mAb pembrolizumab (Keytruda) to our own anti-PD-L1 VHH.
We tested the impact of adding our VHH at different positions on pembrolizumab (Figure 1) and saw that each form retained binding to both PD-1 and PD-L1 targets (Figure 2).
Figure 1: Position of anti-PD-L1 VHH attached to pembrolizumab on both heavy chains (HC) and light chains (LC).
Figure 2: Binding of IgG-VHH fusions to PD-L1 and PD-1 by ELISA. Biotinylated antigens were captured on streptavidin plates. Bound fusion constructs were detected using anti-VHH-HRP.
Overall, the best activity was retained by adding the VHH to the C-terminal of the heavy chain, leaving the most physical space between the PD-1 and PD-L1 binding regions. Given our extensive experience in antibody engineering, we recognize that this is likely to be protein- and target antigen-dependent. Further cellular testing is needed to validate our findings across different applications.
What about IgG-scFv fusions?
Similar IgG-scFv fusions have been in development for over 25 years but have faced limited success and high failure rates in late development or clinical stages. This is likely because it is a challenge to overcome the inherent instability of scFv fragments.
Without the full antibody structure, scFvs rely on non-canonical linkers to maintain VH-VL pairing, but this approach is imperfect, often leading to mispairing and loss of binding. ScFvs also have high hydrophobicity, necessary for VH-VL interaction, which increases aggregation risk, particularly during production and at therapeutic concentrations. These issues require complex expression systems and extensive protein engineering to yield functional proteins.
In contrast, VHHs evolved as naturally stable, single-domain antibodies with a hydrophilic nature. They express efficiently in both prokaryotic (e.g., E. coli, yeast) and eukaryotic systems, are less prone to aggregation, and tolerate extreme pH, temperature, and proteolytic degradation.
Finally scFvs originate from murine antibodies, and so have only ~52% identity with their human counterparts. This means extensive humanization is required, which can cause further stability issues or disrupt target binding. VHHs on the other hand have ~75–90% identity, meaning humanization is more straightforward.
Supercharge your IgG
Although this is only a preliminary study, the benefits of using VHHs for IgG enhancement are clear. VHHs are highly soluble, modular, and can be easily formatted into an IgG-VHH fusion (also known as IgG-dAb or mAb-dAb). These fusions leverage the superior tumor penetration capabilities of VHHs, and are even being explored for crossing the blood-brain barrier (reference).
VHH antibodies show great promise in the biotherapeutics field, not only in adding additional functions to an existing drug, but also as standalone therapeutic agents.
References:
Here at Isogenica, we have more than 20 years of experience in antibody discovery and engineering. If you want to discover how VHHs could supercharge your IgG-based drugs, get in touch with our team at info@isogenica.com.