Bispecific antibodies (bsAbs) represent an important advancement in cancer immunotherapy, enabling dual-targeting mechanisms that enhance tumor specificity, immune activation, and therapeutic efficacy. The structural and functional versatility of bsAbs allows for diverse formats tailored to different clinical needs. As of October 2024, 11 bsAbs have received regulatory approval, seven for hematological malignancies and four for solid tumors. Despite these successes, challenges such as toxicity management and resistance mechanisms remain key areas of investigation.
BsAbs can be broadly categorized into Fc-based (IgG-like and IgG-appended) and fragment-based (IgG-less or IgG-free) molecules. Fc-based bsAbs mirror the natural IgG structure, providing stability and favorable pharmacokinetics (PK). Fragment-based bsAbs are generally smaller, enhancing tumor penetration but often requiring modifications for improved half-life.
Different bsAb formats have been developed to optimize their biological function:
Bispecific T cell engagers (BiTEs): These consist of two single-chain variable fragments (scFvs) connected by a flexible linker. One scFv binds a tumor antigen, while the other binds CD3 on T cells, promoting cytotoxic activity.
Dual-affinity retargeting (DARTs): Featuring a diabody structure with two polypeptide chains, DARTs improve T cell activation and molecular stability.
Tandem diabodies (TandAbs): These tetravalent molecules offer enhanced binding affinity and an extended half-life due to their larger molecular weight.
Bispecific NK cell engagers (BiKEs) and TriKEs: These bsAbs engage NK cells for tumor cell killing, with TriKEs incorporating an IL-15 linker for additional immune stimulation.
Fc-based bsAbs can be engineered using heterodimerization strategies to ensure correct pairing of antibody chains. These include:
Knob-into-hole technology: This method introduces complementary mutations in the CH3 domain to promote heterodimerization.
Fab-arm exchange (FAE): The DuoBody platform mimics the natural Fab exchange process of IgG4 antibodies to generate stable heterodimers.
Charge interactions (e.g., DEKK, ART-Ig, CRIB): Electrostatic forces guide correct heavy chain pairing while preventing homodimer formation.
CrossMab and Wuxibody platforms: These methods swap Fab domains or incorporate alternative constant regions to maintain antigen specificity.
These approaches ensure bsAb functionality, stability, and manufacturability, reducing mispairing risks while maintaining therapeutic efficacy.
Related: Bispecific Antibody Production
The Fc region of bsAbs mediates critical immune functions, such as:
Antibody-dependent cellular cytotoxicity (ADCC) (via FcγRIIIa on NK cells)
Complement-dependent cytotoxicity (CDC) (via C1q binding)
Antibody-dependent cellular phagocytosis (ADCP) (via macrophages)
However, Fc interactions can also cause cytokine release syndrome (CRS) and other immune-related toxicities. Strategies to balance Fc functionality include:
Selecting IgG subclasses: IgG2 and IgG4 have reduced Fcγ receptor binding, minimizing ADCC and CRS.
Engineering Fc mutations: Substitutions such as L234F and N297G reduce FcγR binding to prevent off-target immune activation.
Enhancing ADCC in tumor-targeting bsAbs: Reduced core fucosylation increases FcγRIIIa binding, boosting NK cell-mediated cytotoxicity (e.g., amivantamab, EGFR × cMET, DuoBody).
Optimizing bsAb pharmacokinetics (PK) is another key challenge. Strategies for extending half-life include:
FcRn binding modifications: Mutations like Q311R and M428L enhance FcRn recycling, increasing serum persistence.
Adding Fc regions to fragment-based bsAbs: This extends half-life in BiTEs (HLE-BiTE) and DARTs (DART-Fc).
Serum albumin fusion: Enhances bioavailability and tissue penetration while reducing clearance.
Subcutaneous administration: Provides a sustained-release profile, as seen in trials of subcutaneous blinatumomab.
These modifications improve bsAb stability and bioavailability, reducing dosing frequency and enhancing clinical feasibility.
Related: Applications of Bispecific Antibodies in Therapeutics
BsAbs can be classified based on valency—the number of antigen-binding sites:
Symmetric bsAbs: Typically tetravalent (2+2), providing balanced dual targeting (e.g., cadonilimab, PD-1 × CTLA-4).
Asymmetric bsAbs: Include 1+1 formats (e.g., mosunetuzumab, CD3 × CD20) and 1+2 formats (e.g., glofitamab, CD3 × CD20).
Valency optimization allows for fine-tuned antigen binding, improving tumor targeting and reducing systemic toxicity.
One major concern with bsAbs is on-target off-tumor toxicity, where the antibody binds normal tissues expressing the target antigen. Engineering approaches to mitigate this risk include:
Differential affinity tuning: One antigen-binding arm can have lower affinity to minimize interaction with normal tissues (e.g., TG-1801, CD47 × CD19).
Conditional activation in the tumor microenvironment (TME): Protease-cleavable peptide masks keep bsAbs inactive until tumor-associated enzymes activate them (e.g., TAK-280, CD3 × B7H3).
In trans cell bridging: Requires engagement of both antigens in close proximity, increasing specificity for the TME (e.g., 4-1BB bispecifics targeting tumor-associated antigens).
These approaches enhance bsAb selectivity while minimizing immune-related adverse events.
BsAbs have demonstrated significant clinical efficacy, particularly in hematologic malignancies and select solid tumors. Approved agents include:
Hematologic malignancies: Blinatumomab (CD3 × CD19), mosunetuzumab (CD3 × CD20), and glofitamab (CD3 × CD20).
Solid tumors: Amivantamab (EGFR × cMET) and zanidatamab (HER2 × HER2).
Resistance mechanisms to bsAbs are still under investigation and may involve:
Antigen downregulation or loss: Tumors may reduce expression of the targeted antigens to evade treatment.
TME immunosuppression: Regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and inhibitory cytokines can limit bsAb activity.
Exhaustion of engaged immune cells: Prolonged T cell activation may lead to functional exhaustion, reducing efficacy.
To overcome resistance, rational combination strategies are being explored, including:
BsAbs with immune checkpoint inhibitors: Enhancing T cell persistence and activity.
Sequential therapy with CAR-T cells: Targeting multiple tumor antigens to prevent escape.
TME modulation: Combining bsAbs with agents that alter the immunosuppressive TME.
Ongoing research focuses on further optimizing bsAb formats, improving tumor specificity, and mitigating toxicities. Advances in synthetic biology, computational modeling, and high-throughput screening are expected to accelerate the development of next-generation bsAbs with enhanced safety and efficacy.
With increasing clinical validation, bsAbs are set to become an integral component of cancer immunotherapy, providing novel avenues for precision oncology and combination strategies.
Related: Antibodies to Watch in 2025: Recent Developments in Antibody Therapeutics
At Biointron, we are dedicated to accelerating antibody discovery, optimization, and production. Our team of experts can provide customized solutions that meet your specific research needs, including Bispecific Antibody Production. Contact us to learn more about our services and how we can help accelerate your research and drug development projects.
References:
Herrera, M., Pretelli, G., Desai, J., Garralda, E., Siu, L. L., Steiner, T. M., & Au, L. (2024). Bispecific antibodies: advancing precision oncology. Trends in Cancer, 10(10), 893–919. https://doi.org/10.1016/j.trecan.2024.07.002
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