Bispecific antibodies (bsAbs) enable novel mechanisms of action and/or therapeutic applications that are unattainable with conventional IgG-based antibodies. Bispecific antibodies exist in various formulations targeting different epitopes and exert their anticancer effects through distinct molecular mechanisms. This review provides an overview of the applications of bispecific antibodies in cancer therapy, including: dual modulators mediated by bispecific antibodies; tumor-targeted receptor agonists; bispecific antibody-drug conjugates; bispecific T-cell engagers; activators of natural killer cells and innate immune cells; as well as bispecific checkpoint inhibitors and co-stimulators.

Figure 1: Mechanism of action of bispecific antibodies

1.Dual regulators of signaling pathways:

(1) Dual receptor inhibition:

Many cell surface proteins involved in cellular signal transduction serve as effective targets for antibody-based therapies. Several decades ago, research targeting these proteins—such as the epidermal growth factor receptor (EGFR), HER2, and vascular endothelial growth factor (VEGF)—lay the foundation for the development of nearly all antibody-based therapeutic agents. Although antibody therapies targeting a single, well-defined signaling target demonstrate significant efficacy, disease-related phenotypes are often triggered by multiple signaling pathways acting in concert. Consequently, bispecific antibodies (bsAbs) capable of simultaneously modulating different disease-associated signaling receptors and/or pathways can achieve superior therapeutic outcomes. For example, Amivantamab (JNJ-61186372) targets both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor (MET). Both of these receptors contribute to the proliferation of non-small cell lung cancer (NSCLC); blocking both receptors proves more effective in inhibiting NSCLC growth than blocking either receptor alone.

(2) Ligand–receptor inhibition:

Receptor activation can be inhibited by interfering with receptor dimerization or complex formation, or by blocking the ligand-binding site of the receptor. In cancer, ligands and receptors from multiple complementary or compensatory pathways often collectively promote tumor progression and therapeutic resistance. Bispecific antibodies (bsAbs) can simultaneously block two distinct ligands or simultaneously block a single ligand and its corresponding receptor. Dual targeting of vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG2)—thereby blocking two distinct pathways involved in angiogenesis—has become a central focus of several clinical trials in the field of solid tumors. Vanucizumab is a bispecific antibody targeting both VEGFA and ANG2, which has demonstrated antitumor, anti-angiogenic, and anti-metastatic effects when combined with chemotherapy in preclinical models.

(3) Receptor activation:

Many antitumor responses are mediated by the activation of cell surface receptors, including immune responses or cell death induced by apoptosis. One approach to cancer therapy involves fusing receptor-activating ligands (such as growth factors, cytokines, and (co-)immunostimulatory ligands) with antibodies or antibody fragments to achieve targeted delivery, thereby inducing local or tissue-specific agonism that subsequently triggers cellular responses. An increasing number of such antibody–ligand fusion proteins are entering clinical trials. Additionally, agonistic antibodies can be employed to activate receptors. Bispecific antibodies (bsAbs) targeting specific antigens and members of the TNFRSF family have been demonstrated to function as potent and tumor-selective agonists, mimicking the activity of ligands presented on the cell surface.

2.Bispecific antibody-drug conjugates:

In the field of targeted delivery of payloads such as cytotoxic drugs or radioactive substances using bispecific monoclonal antibodies (bsAbs), two conceptually distinct approaches exist. The first approach is termed "pre-targeted therapy," which utilizes one binding specificity of the bispecific monoclonal antibody to target tumor cells, while the other specificity is employed to subsequently capture the payload on the tumor surface. GD2×DOTA can recognize GD2—a double-sialylated glycolipid highly expressed in cancer—and the radioactive payload 1??Lu-DOTA, thereby assembling and retaining these components within the tumor microenvironment (TME).

The second approach utilizes bispecific antibody-drug conjugates (bsADCs) to deliver a cytotoxic payload to tumor cells while simultaneously targeting two distinct epitopes or two distinct sites on the tumor cell surface, thereby enabling precise targeting of tumor cells.

3.Cancer Immunotherapy:

(1) Immune checkpoint inhibitors (CPIs)

Extending checkpoint inhibitor antibody therapy to bispecific antibodies (bsAbs) may help reduce adverse side effects and enhance the therapeutic efficacy of monoclonal antibody treatments. For example, bispecific antibodies that specifically bind both HER2 and PD-L1 can selectively block PD-L1 activity in tumor cells expressing HER2, yielding superior therapeutic outcomes compared to the use of either component alone.

(2) Effector cell-binding antibodies (including TCEs and ICEs)

Over the past two decades, T-cell-engaging antibodies (TCEs)—which feature the CD3ε chain capable of specifically binding tumor surface antigens and the TCR—have dominated this class of bispecific antibodies (bsAbs). The next-generation TCEs are designed to lack an Fc region; for example, the CD19×CD3ε bispecific T-cell engager (BiTE) blinatumomab was developed based on tandem scFv technology and was initially approved in 2014 for the treatment of B-cell precursor acute lymphoblastic leukemia (B-ALL).

(3) Costimulatory bispecific antibodies

An efficient, durable, and locally specific anti-tumor immune response requires co-stimulatory and co-inhibitory signals to rigorously regulate the activation, differentiation, and maintenance of cytotoxic T cells, natural killer cells, and macrophages. Members of the TNFRSF family (such as 4-1BB, CD40, OX40, TNFRSF18 [also known as glucocorticoid-induced TNFR-related protein, GITR], CD27, or CD30), as well as members of the CD28 immunoglobulin superfamily (such as CD28, CTLA4, PD1, inducible co-stimulatory molecule ICOS, and B and T lymphocyte inhibitory factor BTLA), along with their respective ligands, play a pivotal role in immune regulatory signaling when exerting their local effects through intercellular contact (e.g., between antigen-presenting cells [APCs] and T cells). These receptors provide T cells with what is termed "Signal 2," which can sustain "Signal 1" mediated by the TCR via peptide–MHC–TCR interactions or TCR cross-linking. Targeting these immune regulatory pathways may enhance the efficacy of immunotherapy.

4.brief summary ：

Due to the heterogeneity and adaptive nature of cancer, no single therapeutic approach can serve as a universal immunotherapy for cancer. Instead, antibodies must be custom-designed for specific applications and combined with other therapies to maximize both efficacy and safety. Similar to other anticancer agents, leveraging advanced diagnostic technologies and novel R&D paradigms to introduce bispecific antibodies (bsAbs) and multi-specific antibody-based therapies to patients at an early stage of disease is pivotal to achieving the ultimate goal of curing cancer patients. Tek BioTech (Tianjin) Co., Ltd. has established a comprehensive targeted antibody drug discovery platform utilizing phage display and yeast display technologies. This platform provides global clients with high-quality preliminary candidate sequence screening services, as well as integrated one-stop solutions encompassing downstream bispecific antibody design and preparation, in vitro validation (including various customized validation protocols such as protein-level and cell-level binding assays), and in vivo pharmacodynamic evaluation (via animal model experiments and efficacy assessments), thereby offering robust technical support for clients' research projects.

References ：

[1] Klein, C., Brinkmann, U., Reichert, J.M. et al. The present and future of bispecific antibodies for cancer therapy. Nat Rev Drug Discov 23, 301–319 (2024).

[2] Seimetz, D., Lindhofer, H. & Bokemeyer, C. Development and approval of the trifunctional antibody catumaxomab (anti-EpCAM?×?anti-CD3) as a targeted cancer immunotherapy. Cancer Treat. Rev. 36, 458–467 (2010).

[3] Trinklein, N. D. et al. Eficient tumor killing and minimal cytokine release with novel T-cell agonist bispecific antibodies. mAbs 11, 639–652 (2019).