Monoclonal antibodies (mAbs) have revolutionized modern medicine, offering highly specific targeting across a wide range of diseases, particularly cancer. However, there are some limitations. Tumor antigen heterogeneity, escape mutations, and resistance mechanisms often undermine the long-term effectiveness of single-target therapies. In complex disease settings, relying on a single epitope or pathway is increasingly seen as insufficient.
This is where bispecific antibodies (BsAbs) step in. By binding to two different antigens or epitopes simultaneously, BsAbs offer enhanced precision, greater efficacy, and the potential to overcome resistance. Over the past decade, they have emerged as a powerful next-generation modality – blending the targeting accuracy of traditional antibodies with multifunctional capabilities that pave the way for novel therapeutic strategies.
Figure 1. Differences between monoclonal antibody and bispecific antibody
Key Mechanisms of Bispecific Antibodies
BsAbs are a unique class of engineered antibodies capable of doing that can do more than traditional monoclonal antibodies. By connecting to two different antigens or epitopes at the same time, they help treat diseases in new ways. Here are the main ways BsAbs work:
1. Dual Targeting
Cancer cells often rely on multiple signaling pathways to grow, divide, and survive. Traditional monoclonal antibodies are typically designed to block a single target, such as a specific receptor or growth factor. While effective in some cases, cancer cells can adapt by activating alternative pathways, leading to drug resistance and disease progression. BsAbs, on the other hand, are engineered to simultaneously bind and inhibit two distinct targets involved in tumor growth or survival. By shutting down multiple escape routes at once, BsAbs can deliver a more comprehensive blockade of cancer-promoting signals, making it significantly more difficult for tumor cells to adapt or evade treatment. This dual-action mechanism holds great promise for overcoming resistance and improving treatment outcomes in complex or heterogeneous cancers.
Figure 2. Dual targeting mechanism of BsAbs.
1. Cell bridging
A powerful subclass of bsAbs, known as T-cell engagers, links tumor cells with immune effector cells – most commonly CD3+ T cells. By physically bringing T cells in close proximity to cancer cells, bsAbs facilitate targeted cell killing, independent of the T cell’s native antigen specificity. This mechanism transforms broadly circulating immune cells into highly focused killers, dramatically boosting anti-tumor activity.
Figure 3. Cell bridging mechanism of BsAbs.
1. Functional Modulation
BsAbs can also go beyond simply blocking targets – they can actively change how cells or proteins behave. For example, some BsAbs can force a cancer-related receptor to be internalized and degraded much faster than it normally would, reducing the cell’s ability to receive growth signals. This kind of functional modulation allows BsAbs to fine-tune biological process in ways that traditional drugs often can’t, leading to deeper and more durable anti-tumor effects.
Figure 4. Functional modulation mechanism of BsAbs.
1. Bispecific Antibody Formats
BsAbs come in many shapes and sizes, broadly categorized into IgG-like and non-IgG-like formats. Each class has unique features suited for different therapeutic needs.
- IgG-like BsAbs These resemble traditional antibodies and retain the Fc region, which enables functions like antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody recycling (longer half-life). A number of engineering strategies – some of which are highlighted below - have been developed to ensure correct pairing of the two different antibody arms.
- Quadroma: Fuses two different hybridoma cells to produce a bispecific antibody in a single cell. Simple but results in mixtures of antibody species, requiring purification to remove unwanted combinations.
- Duobody: Uses controlled Fab-arm exchange between two parental antibodies under redox conditions to promote heterodimer formation. This in-vitro process preserves Fc function and structural integrity while simplifying production.
- Knobs-into-Holes (KiH): Engineers CH3 domain to create a “knob” and a “hole” on opposite heavy chains, promoting correct Fc heterodimerization. Solves heavy chain mispairing but often combined with other methods to address light chain mispairing.
- CrossMab: Swaps domains with one Fab arm (e.g., CH1 with CL, VH with VL) to ensure correct light-heavy chain pairing, often used with KiH to promote heavy chain heterodimerization. Widely adopted with multiple BsAbs in clinical development.
- SEEDbody: Engineered CH3 domains comprising alternating human IgG/IgA segments drive Fc heterodimerization, preserving Fc function while enabling asymmetric fusion formats for bsAbs.
- Dual-Variable Domain Ig (DVD-Ig):Tandemly stacks two different VH-VL pairs on each chain, separated by linkers, allowing one IgG-like molecu
- le to bind two targets without chain-pairing issues. Retains Fc function.
- IgG-scFv: Fuses a single-chain variable fragment (scFv) to the IgG framework, typically at the C-terminus, enabling bispecificity while retaining Fc-mediated functions and extended half-life.
- Κλ-body: Utilizes natural pairing preferences of κ and λ light chains to promote correct light-heavy chain assembly in bispecific antibodies, reducing light chain mispairing without extensive engineering.
Half-Molecule Exchange: Leverages the natural tendency of IgG4 antibodies to exchange half molecules (one heavy chain and its paired light chain), enabling bispecific antibody formation with preserved Fc functions and simplified production.
Figure 5. Molecular formats of IgG-like bispecific antibodies
2. 2. Non-IgG-like BsAbs
These antibody formats are generally smaller and often lack the Fc region, resulting in improved tissue and tumor penetration as well as faster systemic distribution. Due to the absence of Fc, they typically have shorter serum half-lives but exhibit high modularity and flexibility, making them well-suited for immune cell engagement and rapid action.
ScFv-based BsAbs
These bispecific antibodies are constructed primarily from single-chain variable fragments (scFvs). Common formats include:
- Tandem scFv: Two scFvs linked in tandem to bind two different antigens. One representative example is Bispecific T-cell engager (BiTEs), which specifically engage T cells via CD3, recruiting them to tumor cells.
- Dual-Affinity Re-Targeting Molecules (DARTs): Engineered to improve stability and affinity over traditional tandem scFvs.
- Diabody and TandAbs: These are multimeric assemblies of scFvs forming dimeric or tetrameric structures, increasing avidity and enabling dual targeting.
Other non-IgG-like BsAbs
- Nanobodies: Small, stable single-domain antibodies from camelids, offering excellent tissue penetration but usually lacking Fc, leading to shorter half-life.
- Dock-and-Lock (DNL): Modular system using specific protein domain interactions to assemble stable bispecific complexes with flexible design.
- scFv-HSA-scFv: A design that inserts human serum albumin (HAS) between two scFvs to extend the molecule’s serum half-life, combing the targeting flexibility of scFvs with the long circulation time of HAS.
Figure 6. Molecular formats of non-IgG-like bispecific antibodies
Conclusion
Bispecific antibodies offer the unique advantages of simultaneously targeting two different antigens or epitopes, addressing challenges like tumor heterogeneity and drug resistance. Their broad range of IgG-like and non-IgG-like formats provides versatile therapeutic strategies tailored to different clinical needs.
Moreover, this dual-targeting capability lays the groundwork for bispecific antibody-drug conjugates, which can enhance tumor specificity and improve drug delivery, offering a promising next-generation of ADCs.
Reference
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