Bispecific antibodies (bsAbs) represent an evolution in antibody engineering. Unlike conventional monoclonal antibodies that bind to a single epitope, bsAbs are designed to engage two distinct targets simultaneously. This dual-binding capability has opened numerous opportunities in immunotherapy, particularly for cancer treatment, where one arm can bind to a tumor-associated antigen while the other recruits or activates immune effector cells, such as T cells or NK cells.
While first-generation bsAbs have shown promising clinical potential, they face several challenges in their therapeutic application. The structural complexity that enables dual targeting also introduces issues with stability, aggregation, and solubility. In addition, off-target effects and immunogenicity remain major concerns in advancing bsAbs safely into clinical settings.
To address these limitations, researchers are now turning to conditionally active bsAbs that only bind or activate their targets under specific conditions. This might be a particular pH or the presence of a specific molecule.
Conditional activation allows the bsAb to remain inactive in healthy tissues and only become active in the tumor microenvironment, potentially improving specificity, efficacy, and safety.
Case study in conditional CD137 agonism
An example of this approach comes from Zhang and colleagues, who engineered a c-MET x CD137 bsAb designed for conditional CD137 agonism in cancer immunotherapy.1 Their work showed that the bsAb achieved tumor specific immune activation while minimizing off-target effects.
This work is particularly relevant as two anti-CD137 agonistic antibodies, urelumab and utomilumab, have demonstrated promising therapeutic potential but are associated with dose-limiting systemic toxicities and modest clinical efficacy, respectively, highlighting the importance of strategies that enable more targeted immune activation.
The bsAb was engineered to bind c-MET, a tumor-associated receptor, with a monovalent binder to avoid unwanted activation of tumor-promoting pathways while also conferring trivalent CD137 engagement. This only occurred when the bsAb was anchored to the tumor cell surface via c-MET, enabling localized immune activation.
This design led to strong T cell activation and cytokine release, but only in the presence of both targets, demonstrating how conditional agonism can balance potency with safety. Crucially, when the team combined the bsAb with an anti-PD-1 agent, Pembrolizumab, they observed higher levels of target-induced T cell cytokine release compared to either treatment alone, indicating a superior enhancement of target induced T cell cytokine release.
Functional assays enabling precise characterization
To support this rational design, the team used Revvity's AlphaLISA™ and AlphaLISA™ SureFire® Ultra™ (ALSU) immunoassay kits to evaluate signaling events and cytokine production. These homogeneous, high-sensitivity assays were ideal for evaluating phosphorylation of key signaling proteins and detecting immune activation markers like Granzyme B and IFN-γ. Paired with the EnVision™ plate reader, the platform enabled rapid, reproducible analysis across multiple bsAb constructs.
Read the full story
Do you want to learn more about how conditional bsAb strategies are shaping the future of cancer immunotherapy? Read the full literature review on c-MET x CD137 bsAb development.
Reference
- Zhang, H., Wang, Q., Yalavarthi, S., Pekar, L., Shamnoski, S., Hu, L., Helming, L., Zielonka, S., & Xu, C. (2024). Development of a c-MET x CD137 bispecific antibody for targeted immune agonism in cancer immunotherapy. Cancer Treatment and Research Communications, 39, 100805. https://doi.org/10.1016/j. ctarc.2024.100805
For research use only. Not for use in diagnostic procedures.
