2023 Impact Factor
The advent of antibody therapy has profoundly transformed modern medicine. The concept of using antibodies for therapeutic purposes dates to the late 19th and early 20th centuries (von Behring, 2024). The introduction of hybridoma technology in the 1970s enabled the production of monoclonal antibodies (mAbs) and facilitated extensive research on disease treatment (Kohler and Milstein, 1975). However, mAbs have not sufficiently addressed drug resistance in numerous diseases, particularly where tumor biology or underlying mechanisms are not fully understood (Aldeghaither
The first clinically successful BsAb, catumaxomab (Removab®), was approved by the European Medicines Agency (EMA) in 2009 (withdrawn in 2017) for the treatment of malignant ascites in patients with epithelial cell adhesion molecule (EpCAM)-positive cancers (Tiller and Tessier, 2015). Since then, numerous BsAbs that target various diseases have demonstrated significant success in clinical development. BsAbs offer several advantages, particularly in oncology, where they enhance therapeutic efficacy by recruiting immune cells to cancer cells through simultaneous targeting of two distinct antigens. Additionally, because tumors often develop resistance to therapies targeting a single antigen, BsAbs have the potential to overcome this resistance by simultaneously targeting multiple antigens. They can also target multiple pathways to achieve effects similar to those of combination therapies, potentially leading to synergistic effects and improved clinical outcomes (Zhu
BsAbs have the potential to improve therapeutic efficacy, resistance and target specificity over conventional mAbs (Zhu
Variable regions of antibodies composed of variable heavy (VH) and variable light (VL) chains constitute the antigen-binding sites (Samuel and Naz, 2008). These regions are responsible for the specificity and affinity of antibodies toward their respective antigens (Sela-Culang
Table 1 Structural designs and clinical indications of bispecific antibodies
Structural design | Drug name (trade name) | Format | Targets | Mode of action | Indication | Approval status |
---|---|---|---|---|---|---|
IgG | Emicizumab (Hemlibra®) | Common light chain | Factor IXa, Factor X | Co-factor for blood clotting | Hemophilia A | 2017 (FDA) |
Amivantamab (Rybrevant®) | Duobody | EGFR, MET | Dual inhibition of EGFR and MET | NSCLC with EGFR exon 20 insertion mutation | 2021 (FDA) | |
Faricimab (Vabysmo®) | CrossMab | VEGF-A, ANG-2 | Dual inhibition of VEGF and ANG-2 | Neovascular AMD | 2022 (FDA) | |
Teclistamab (Tecvayli®) | Duobody | BCMA, CD3 | T cell engager | Multiple myeloma | 2022 (FDA) | |
Mosunetuzumab (Lunsumio®) | Knobs-into-holes | CD20, CD3 | T cell engager | Relapsed/refractory non-Hodgkin lymphoma | 2022 (FDA) | |
Epcoritamab (Epkinly®) | Duobody | CD20, CD3 | T cell engager | Relapsed/refractory diffuse large B-cell lymphoma | 2023 (FDA) | |
Glofitamab (Columvi®) | CrossMAb | CD20, CD3 | T cell engager | Relapsed/refractory diffuse large B-cell | 2023 (FDA) | |
Talquetamab (Talvey®) | Duobody | GPRC5D, CD3 | T cell engager | Multiple myeloma | 2023 (FDA) | |
Elranatamab (Elrexfio®) | Modified IgG | BCMA, CD3 | T cell engager | Multiple myeloma | 2023 (FDA) | |
Cadonilimab (Kaltanni®) | Tetrabody | PD-1, CTLA-4 | Dual immune checkpoint inhibition | Relapsed/refractory cervical cancer | 2023 (NMPA) | |
Catumaxomab (Removab®) | Triomab | EpCAM, CD3 | T cell engager, Fc-mediated effects | Ovarian ascites | 2009 (Withdrawn) | |
Non-IgG | Blinatumomab (Blincyto®) | BiTE | CD19, CD3 | T cell engager | B cell precursor ALL | 2014 (FDA) |
Tebentafusp (Kimmtrak®) | ImmTAC | gp100, CD3 | T cell engager | Uveal melanoma | 2022 (FDA) |
IgG-like BsAbs closely resemble conventional IgG antibodies but are engineered to possess dual specificity (Krishnamurthy and Jimeno, 2018). The Duobody platform utilizes a precise process in which two IgG1 molecules are individually engineered and recombined to generate bispecific antibodies. Each IgG1 molecule is independently engineered with specific mutations in the CH3 domain to facilitate correct chain pairing and association. After production and purification, the two IgG1 molecules undergo a controlled Fab-arm exchange, resulting in the formation of a bispecific antibody with high efficiency and yield (Labrijn
First, emicizumab (Hemlibra®) is modeled on the structure of a human IgG antibody, maintaining the typical Y-shaped configuration. The Fab regions are engineered to bind to different antigens that target the factors IXa and X. The Fc region remains unmodified, providing an extended half-life through interaction with the neonatal Fc receptor for IgG (FcRn) while minimizing immune-mediated clearance (Kitazawa
In summary, IgG-like BsAbs represent a class of therapeutic agents that retain their structural stability and functional properties while simultaneously targeting multiple antigens. These antibodies, with their optimized design and engineering, hold significant promise in precision medicine, particularly for the treatment of complex diseases such as cancer. Further research should focus on expanding the clinical applications of BsAbs, exploring strategies to enhance their safety and efficacy, and ensuring their effective integration into therapeutic regimens.
Non-IgG-like BsAbs have a more compact and flexible structure than IgG-like BsAbs. One strategy involves dimerizing single-chain variable fragments (scFvs) by incorporating a peptide linker between the two domains, resulting in the formation of two antigen-binding sites oriented in opposite directions (Holliger
BsAbs demonstrate enhanced efficacy by simultaneously targeting multiple antigens, thereby potentially overcoming the limitations of single-target therapies. However, these trials have revealed challenges in clinical applications, including the induction of immune responses and stability-related issues. Non-IgG-like BsAbs, which generally exhibit a smaller size and a more flexible structure than IgG-like antibodies, may exhibit lower immunogenicity owing to improved tissue penetration (Fan
The MOA of BsAbs is diverse and depend on their specific design and target (Sun
One of the most prominent applications of BsAbs is in cancer immunotherapy where they are used to redirect and activate T cells against tumor cells (Qi
TCEs simultaneously bind to both tumor antigens and T cells. One arm of the TCE is designed to specifically bind to a tumor-associated antigen (TAA) expressed on the surface of cancer cells. Antigens such as CD19, CD20, B cell maturation antigen (BCMA), CD123, human epidermal growth factor receptor 2 (HER2), or EGFR, are often overexpressed in tumor cells and serve as markers for TCE targeting (Baeuerle and Wesche, 2022; Cattaruzza
Upon simultaneous binding to the tumor antigen and the CD3 subunit on T cells, TCE facilitates the formation of stable immunological synapses (Baeuerle and Wesche, 2022). This synapse brings T and cancer cells into proximity, creating an environment conducive to T cell activation (Strohl and Naso, 2019). Engagement of CD3 triggers T cell activation, leading to a cascade of intracellular signaling events that results in T cell proliferation and cytokine secretion (Smith-Garvin
In addition to cytotoxicity, activated T cells secrete cytokines, including interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), which further enhance the immune response by recruiting additional immune cells and promoting inflammation at the tumor site (Burkholder
Several TCEs have been approved for clinical use, demonstrating their effectiveness in the treatment of various types of cancer: mosunetuzumab (Kang, 2022a), epcoritamab, glofitamab (Dickinson
Immune checkpoints are regulatory mechanisms in the immune system that are crucial for maintaining self-tolerance and for adjusting the duration and amplitude of physiological immune responses in peripheral tissues, thereby reducing collateral tissue damage (Pardoll, 2012; Taefehshokr
BsAbs can be designed to simultaneously target two distinct immune checkpoints: PD-1 and CTLA-4. This approach was designed to overcome the limitations of therapies that target a single checkpoint and provide more comprehensive activation of the immune system (Farhangnia
BsAbs simultaneously inhibit two distinct molecular pathways involved in disease progression, thereby offering a broader and more effective therapeutic approach (Huang
BsAbs bind and neutralize soluble ligands, preventing them from interacting with their receptors, and thereby modulating disease processes (Kontermann, 2012). A prime example of this dual ligand targeting capability is faricimab, an FDA-approved BsAb. Faricimab is specifically designed to simultaneously bind and inhibit both ANG-2 and VEGF-A, two key players in pathological angiogenesis (Shirley, 2022). It neutralizes VEGF-A to prevent it from promoting abnormal blood vessel growth and leakage, while also inhibiting ANG-2, which competes with angiopoietin-1 (ANG-1) to bind to Tie-2 receptors (Shirley, 2022). This inhibition helps restore vascular stability and reduce inflammation by allowing ANG-1 to effectively activate the Tie-2 receptor (Joussen
BsAbs also act as enzyme or cofactor mimics, facilitating reactions by bringing enzymes and substrates together or by substituting missing or dysfunctional cofactors in enzymatic pathways (Kang
During drug development, PK involves absorption, distribution, metabolism, and excretion (ADME) in the body. PK studies are essential for predicting a drug’s blood concentration, duration of action, systemic distribution, cost reduction, and personalized treatment (Meibohm and Derendorf, 2002; Reichel and Lienau, 2016; Kantae
Table 2 Pharmacokinetic profiles of bispecific antibodies
Drug name (trade name) | Absorption | Volume of distribution | Clearance | Half-life | Ref. |
---|---|---|---|---|---|
Blinatumomab (Blincyto®) | Intravenous | 5.27 L | 3.11 L/h | ~2.1 h | Food and Drug Administration, 2024c |
Emicizumab (Hemlibra®) | Subcutaneous | 10.4 L | 0.24 L/day | ~30 days | Food and Drug Administration, 2024e |
Amivantamab (Rybrevant®) | Intravenous | 5.34 L | 360 ± 144 mL/day | ~13.7 days | Food and Drug Administration, 2024b |
Tebentafusp (Kimmtrak®) | Intravenous | 7.56 L | 16.4 L/day | ~7.5 h | Food and Drug Administration, 2024i |
Faricimab (Vabysmo®) | Intravitreal | 5.91 L | Not available | ~12.0 days | Food and Drug Administration, 2024a |
Teclistamab (Tecvayli®) | Subcutaneous | 5.63 L | 0.472 L/day | ~15 days | Food and Drug Administration, 2024j |
Mosunetuzumab (Lunsumio®) | Intravenous | 5.49 L | 1.08 L/day | ~16.1 days | Food and Drug Administration, 2024g |
Epcoritamab (Epkinly®) | Subcutaneous | 25.6 L | 0.53 L/day | ~33.0 days | Food and Drug Administration, 2024f |
Glofitamab (Columvi®) | Intravenous | 5.61 L | 0.674 L/day | ~7.6 days | Food and Drug Administration, 2024d |
Talquetamab (Talvey®) | Subcutaneous | 10.1 L | 0.90 L/day | ~17.7 days | Food and Drug Administration, 2024h |
Elranatamab (Elrexfio®) | Subcutaneous | 7.76 L | 0.324 L/day | ~22 days | Food and Drug Administration, 2023 |
Cadonilimab (Kaltanni®) | Subcutaneous | 6.1 L | Not available | ~4.76 days | Keam, 2022; Li |
Catumaxomab (Removab®) | Intraperitoneal | Not specified | Not available | ~2.13 days | Ruf |
Table 3 Binding affinity and protein binding properties of bispecific antibodies
Drug name (trade name) | Target 1 | Target 1 (Kd) | Target 2 | Target 2 (Kd) | Plasma protein binding (%) | Manufacturer |
---|---|---|---|---|---|---|
Blinatumomab (Blincyto®) | CD19 | 1.5×10–9 | CD3 | 2.6×10–7 | 5 | Amgen Inc. |
Emicizumab (Hemlibra®) | FIXa | 1.5×10–6 | FX | 9×10–7 | 97 | Roche |
Amivantamab (Rybrevant®) | EGFR | 1.4×10–9 | MET | 4×10–11 | 90 | Johnson & Johnson |
Tebentafusp (Kimmtrak®) | gp100 | 2.4×10–11 | CD3 | 3.8×10–8 | 85 | Immunocore |
Faricimab (Vabysmo®) | VEGF | 2×10–8 | Ang-2 | 1×10–9 | 75 | Roche |
Teclistamab (Tecvayli®) | BCMA | 1.8×10–10 | CD3 | 2.8×10–8 | 80 | Johnson & Johnson |
Mosunetuzumab (Lunsumio®) | CD20 | 6.8×10–8 | CD3 | 4×10–8 | 78 | Roche |
Epcoritamab (Epkinly®) | CD20 | 2.4×10–9 | CD3 | 4.7×10–9 | 95 | Genmab A/S |
Glofitamab (Columvi®) | CD20 | N/A | CD3 | 4×10–10 | 77 | Roche |
Talquetamab (Talvey®) | GPRC5D | 4.21×10–9 | CD3 | 2.5×10–8 | 70 | Johnson & Johnson |
Elranatamab (Elrexfio®) | BCMA | 3.8×10–11 | CD3 | 1.7×10–9 | 70 | Johnson & Johnson |
Cadonilimab (Kaltanni®) | PD-1 | 1×10–10 | CTLA4 | 4.1×10 | 60 | Akeso Inc. |
Catumaxomab (Removab®) | EpCAM | 5.6×10–10 | CD3 | 4.4×10–9 | 70 | Fresenius Biotech |
The absorption route significantly influences bioavailability. BsAbs have poor stability in the gastrointestinal tract and low permeability across the gut wall, and consequently negligible oral bioavailability, similar to conventional therapeutic proteins. BsAbs can be administered via intravenous (IV), intraperitoneal (IP), or subcutaneous (SC) injection (Rathi and Meibohm, 2015). Although BiTE molecules are small, which may suggest rapid absorption kinetics, they are typically administered intravenously to achieve more rapid systemic exposure. IgG-like BsAbs, such as CrossMAbs, are commonly administered by SC injection due to their relatively large molecular size, which leads to slower but sustained absorption. This difference in profile between the two platforms can significantly influence the onset of therapeutic action and the overall duration of treatment. For example, the T cell engager blinatumomab is administered intravenously, providing rapid systemic availability, whereas elranatamab is administered subcutaneously, allowing for sustained absorption. IV administration delivers the drug directly into the bloodstream, thereby ensuring its immediate and complete bioavailability. This characteristic is particularly beneficial for BsAbs that require rapid therapeutic action, such as amivantamab, which is used to treat non-small cell lung cancer (NSCLC). Through this route, amivantamab quickly reaches the systemic circulation, enabling prompt engagement with its target antigens, EGFR and mesenchymal-epithelial transition factor (MET) (Cho
In PK, the volume of distribution (Vd) is a critical parameter that provides insights into the distribution characteristics of a drug (Gillette, 1973). Higher Vd values indicate that the drug extensively permeates tissues and extracellular fluids, which is particularly important for drugs targeting widespread or deep-seated tissues such as tumors.
The differences in Vd among the BsAbs can be attributed to several complex factors. First, the molecular structure and size of each BsAb play crucial roles in determining the distribution of the drug beyond the central compartment. For BsAbs with a relatively large molecular size, their movement from the blood vessels to the interstitial tissue space occurs mainly convection rather than diffusion (Baxter
Antigen specificity and target tissue distribution are also critical determinants of Vd. BsAbs targeting antigens that are primarily confined to the bloodstream or lymphatic system generally exhibit lower Vd values, whereas those targeting antigens with a broader tissue distribution tend to show higher Vd values, which can be attributed to increased tissue penetration. BsAbs that target cell surface antigens on B cell lymphoma and T cell, such as blinatumomab (BiTE, 55 kDa), typically have lower Vd values (5.27 L), indicating limited tissue distribution. Similarly, mosunetuzumab and glofitamab, bispecific antibodies with comparable pharmacokinetic characteristics, have Vd values of 5.49 L and 5.61 L, respectively, further supporting this distribution pattern (Food and Drug Administration, 2024b). In contrast, epcoritamab, which targets tissue-bound antigens, has a higher Vd (25.6 L) and a broader tissue distribution (Food and Drug Administration, 2024f). To optimize the uptake and penetration of BsAbs into solid tumors, several factors need to be carefully evaluated, including the appropriate binding affinities for tumor antigens and effector cells, as well as the optimal molecular size to achieve sustained systemic exposure (Chen and Xu, 2017).
The physicochemical properties of a drug, including lipophilicity, charge, and stability, play crucial roles in its distribution. For instance, lipophilic drugs are more likely to traverse cell membranes and accumulate in tissues, resulting in higher Vd, whereas hydrophilic drugs are more likely to remain confined to the vascular space (Liu
These factors are crucial for optimizing the pharmacokinetic profiles of BsAbs, enabling precise adjustment of dosing regimens to maximize therapeutic efficacy while minimizing adverse effects. The observed variability in Vd among BsAbs highlights the necessity for individualized PK evaluation during drug development and clinical applications. This approach ensures that each therapeutic agent is optimally deployed to achieve maximum efficacy across patient populations.
BsAbs undergo catabolism primarily via proteolytic enzymes in the reticuloendothelial system, particularly in the liver and kidneys (Vugmeyster
Advanced engineering strategies such as CrossMab and knobs-into-holes aim to enhance the structural stability and circulation time of BsAbs. CrossMab technology involves the exchange of domains between the heavy and light chains to maintain stability and functionality (Klein
Like conventional mAbs, BsAbs may undergo target-mediated drug disposition (TMDD) as clearance pathways. At lower doses, BsAbs that bind to cell membrane antigens may exhibit dose-dependent clearance, leading to faster elimination and shorter half-life. At higher doses, when the target-mediated clearance is saturated, PK become linear. In a xenograft mouse model, systemic bioavailability of catumaxomab decreased with increasing EpCAM-positive tumor burden and CD3-positive cells, indicating target-mediated disposition (Ruf
Understanding the metabolic pathways of BsAbs is critical to optimize their therapeutic potential. Advanced engineering strategies are being developed to enhance these properties and improve the clinical efficacy of BsAbs. This variability in metabolic pathways highlights the necessity for individualized PK assessments during drug development to ensure that each therapeutic agent is effectively utilized across diverse patient populations.
The contribution of renal clearance to BsAb elimination is considered to be influenced by the molecular size. For BsAbs with molecular weights below 70 kDa such as BiTE and DART, which fall under the renal filtration threshold, renal clearance may contribute significantly to the overall clearance and leading to short systemic persistence. BiTE molecules (approximately 55 kDa) are generally excreted more rapidly via the kidneys due to their small size and rapid clearance, whereas IgG-like BsAbs, including CrossMAbs, are excreted more slowly due to their longer half-life and FcRn recycling mechanisms (Chen and Xu, 2017). This results in less frequent dosing for IgG-like BsAbs, compared to BiTEs, which require more frequent dosing to maintain therapeutic efficacy. PK parameters, such as half-life, provide insights into the duration of action and dosing frequency required to maintain therapeutic levels. Blinatumomab (a small BsAb in BiTE) exhibits a short half-life of approximately 2.1 h in human, probably due to its small molecular size and lack of an Fc domain (Food and Drug Administration, 2024c). This rapid clearance necessitates continuous IV infusion to maintain therapeutic efficacy (Liu, 2018). Tebentafusp (a small BsAb in ImmTAC) also has a short half-life of approximately 7.5 h in human, probably due to its small molecular size of 77 kDa and lack of an Fc domain (Food and Drug Administration, 2024i). In contrast, cadonilimab is an anti-PD-1/CTLA-4 BsAb in tetrabody format with a molecular weight of approximately 200 kDa (Keam, 2022). The half-life of cadonilimab is approximately 4.76 days (Keam, 2022). Emicizumab (approximately 145 kDa) has a notably prolonged half-life of approximately 30 days, which facilitates less frequent dosing regimens and enhances patient compliance and convenience (Food and Drug Administration, 2024e). This extended half-life was attributed to the FcRn-mediated recycling process, which mirrors that of endogenous IgG, allowing sustained therapeutic levels with less frequent administration (Liu, 2018; Schmitt
These examples highlight the impact of molecular design on the PK and excretion patterns of BsAbs. For instance, molecules such as faricimab and glofitamab, which are engineered with Fc regions, benefit from FcRn-mediated recycling, leading to reduced clearance rates (Nicolo
Advancements in BsAbs have marked pivotal developments in therapeutic antibody engineering, providing enhanced efficacy and specificity relative to conventional mAbs. Through simultaneous targeting of two distinct antigens, BsAbs can overcome the challenges frequently encountered with mAbs, including drug resistance and incomplete target coverage. The MOA of BsAbs, which enables the direct engagement of immune cells with cancer cells and the inhibition of multiple signaling pathways, offers a more targeted therapeutic approach for treating complex diseases. Nevertheless, the sophisticated structure and dual-targeting capability of BsAbs introduce distinct PK challenges that require comprehensive investigation to optimize their design and clinical utility. As engineering refinements continue to address these challenges, future research should focus on broadening the clinical applicability of BsAbs, enhancing their safety profiles, and developing strategies for their seamless integration into combination therapies. Continued innovation in BsAb technology has the potential to deliver novel therapeutic strategies for conditions that are currently challenging to treat with existing modalities, thereby significantly advancing the field of precision medicine.
This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (Nos. 2022R1A2C1004714, 2021R1A5A2031612, and 2024M3A9J4006509). S. M. Choi was supported by a grant (21153MFDS602) from the Ministry of Food and Drug Safety.
The authors declare no conflict of interest.