Abstract
Bispecific antibodies (BsAbs) represent a rapidly evolving class of immunotherapeutic agents engineered to simultaneously bind two distinct targets, thereby mediating novel functionalities beyond the capabilities of conventional monoclonal antibodies. This report provides a comprehensive overview of BsAb technology, encompassing their diverse mechanisms of action, structural formats, clinical applications (with a focus on cancer immunotherapy), safety considerations, and future directions. We delve into the intricate details of BsAb engineering, examining various formats designed to optimize stability, effector function, and pharmacokinetic properties. Furthermore, we critically analyze the clinical trial data across different cancer types, highlighting both successes and challenges encountered in BsAb development. A key focus is placed on the comparative advantages and disadvantages of BsAbs relative to other immunotherapeutic modalities, such as CAR T-cell therapy and immune checkpoint inhibitors. Finally, we explore emerging trends and future perspectives in the field, including the development of multi-specific antibodies, novel target combinations, and strategies to overcome resistance mechanisms, positioning BsAbs as a pivotal component of the evolving cancer treatment landscape.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
1. Introduction
Monoclonal antibodies (mAbs) have revolutionized cancer therapy, demonstrating significant clinical benefits in a wide range of malignancies. However, the inherent specificity of mAbs limits their functional versatility. Bispecific antibodies (BsAbs) overcome this limitation by enabling simultaneous engagement of two distinct targets, offering a broad spectrum of therapeutic possibilities. This dual-targeting capability allows BsAbs to bridge immune cells to tumor cells, block multiple signaling pathways, enhance receptor internalization, and deliver payloads directly to the tumor microenvironment. The initial development of BsAbs faced significant challenges related to production, stability, and immunogenicity. However, advancements in antibody engineering have yielded a plethora of formats with improved pharmacokinetic (PK) and pharmacodynamic (PD) properties, paving the way for clinical translation. The first BsAbs approved for clinical use, such as blinatumomab, targeted hematological malignancies, demonstrating the potential of this technology to redirect T cells against cancer cells. Subsequent approvals and ongoing clinical trials highlight the expanding role of BsAbs in diverse cancer settings. This report provides a comprehensive overview of BsAb technology, focusing on their mechanisms of action, clinical applications, safety profiles, and future directions.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
2. Mechanisms of Action
BsAbs exhibit a diverse range of mechanisms of action, broadly categorized as follows:
2.1 T-Cell Redirection
The most extensively studied mechanism involves the recruitment and activation of T cells to eliminate tumor cells. These BsAbs typically bind to a T-cell surface marker (e.g., CD3) and a tumor-associated antigen (TAA). The interaction between the BsAb and CD3 activates the T cell, leading to the formation of a cytotoxic synapse with the tumor cell. This synapse facilitates the release of cytotoxic granules, such as perforin and granzymes, which induce apoptosis in the tumor cell. Mosunetuzumab, epcoritamab, and glofitamab, mentioned in the introduction, are prime examples of CD3-engaging BsAbs targeting CD20 on lymphoma cells. The strength of the CD3 binding affinity is a critical factor in determining the efficacy and toxicity of these BsAbs. Overly potent CD3 binding can lead to excessive T-cell activation and cytokine release syndrome (CRS), while insufficient binding may result in suboptimal T-cell activation and tumor cell killing. A fine balance is therefore required when designing these types of therapies. Further, the choice of TAA is also crucial, as it determines the specificity of the T-cell redirection. The ideal TAA should be highly expressed on tumor cells but minimally expressed on normal tissues to minimize off-target toxicity.
2.2 Immune Checkpoint Blockade
Immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-CTLA-4 antibodies, have revolutionized cancer therapy by unleashing the power of the immune system. BsAbs can be engineered to simultaneously block two or more immune checkpoints, potentially overcoming resistance mechanisms and enhancing anti-tumor immunity. For example, a BsAb targeting both PD-1 and CTLA-4 could provide synergistic blockade of these inhibitory pathways, leading to more robust T-cell activation. The advantage of a bispecific checkpoint inhibitor, compared to a combination of two separate monoclonal antibodies, is to promote T-cell activation specifically within the tumour microenvironment by co-locating both immune checkpoint inhibitors to the tumour. BsAbs that block PD-L1 and TGF-β are also under development, as TGF-β is known to promote immune suppression in the tumor microenvironment. The simultaneous blockade of these two pathways can enhance T-cell infiltration and cytotoxicity. One potential disadvantage of bispecific checkpoint inhibitors is the enhanced risk of immune-related adverse events (irAEs). Therefore, careful monitoring and management of irAEs are essential in clinical trials.
2.3 Receptor Clustering and Internalization
BsAbs can induce receptor clustering and internalization, leading to the downregulation of oncogenic signaling pathways. For example, a BsAb targeting two different epitopes on the epidermal growth factor receptor (EGFR) could induce receptor internalization and degradation, thereby inhibiting EGFR signaling. This approach is particularly attractive for overcoming resistance to EGFR inhibitors. Similarly, BsAbs can induce the internalization of death receptors, such as TRAIL-R1 and TRAIL-R2, leading to apoptosis in tumor cells. The advantage of this approach is that it can directly kill tumor cells, independent of T-cell activation. However, the efficacy of this approach may be limited by the expression levels of the target receptors and the sensitivity of tumor cells to apoptosis.
2.4 Delivery of Therapeutic Payloads
BsAbs can be used to deliver therapeutic payloads, such as chemotherapy drugs, toxins, or radioisotopes, directly to tumor cells. This approach can enhance the efficacy of these agents while minimizing systemic toxicity. For example, a BsAb targeting a tumor-associated antigen and a receptor that internalizes upon binding could deliver a cytotoxic payload specifically to tumor cells. The challenge with this approach is the complexity of conjugating the payload to the BsAb without affecting its binding affinity or stability. Furthermore, the payload must be released inside the tumor cell to exert its cytotoxic effect. Advances in linker technology have improved the delivery of therapeutic payloads and reduced off-target toxicity.
2.5 Bridging Effector Cells to Tumor Cells
Beyond T cells, BsAbs can recruit and activate other effector cells, such as natural killer (NK) cells and macrophages, to eliminate tumor cells. NK cells express the FcγRIIIa receptor, which binds to the Fc region of IgG antibodies. A BsAb targeting a tumor-associated antigen and FcγRIIIa can bridge NK cells to tumor cells, leading to NK cell activation and antibody-dependent cell-mediated cytotoxicity (ADCC). Similarly, BsAbs can activate macrophages to phagocytose tumor cells through antibody-dependent cellular phagocytosis (ADCP). The choice of effector cell depends on the specific tumor type and the desired mechanism of action. For example, NK cell-mediated cytotoxicity may be more effective for tumors that express low levels of MHC class I molecules, which are less susceptible to T-cell killing. An advantage of using NK cells over T cells is they do not require pre-sensitisation and do not cause as severe cytokine release syndrome.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
3. BsAb Formats and Engineering
BsAbs can be produced using various formats, each with its own advantages and disadvantages. The first-generation BsAbs were typically produced by chemical conjugation or quadroma technology, which resulted in heterogeneous mixtures of antibody species. These methods have been largely replaced by recombinant DNA technology, which allows for the precise control of antibody structure and function. Some commonly used BsAb formats include:
3.1 IgG-like BsAbs
These BsAbs retain the overall structure of a conventional IgG antibody but have modifications in the variable regions to confer bispecificity. One approach is the “knobs-into-holes” technology, which introduces mutations in the CH3 domains of the two heavy chains to promote heterodimerization. This technology has been used to produce BsAbs with improved stability and reduced aggregation. Another approach is the use of common light chains, which pair with different heavy chains to generate bispecificity. Several IgG-like BsAb formats have been approved for clinical use, including emicizumab for hemophilia A and REGN-EB3 for Ebola virus infection. These formats typically have longer half-lives and better effector function than smaller BsAb formats.
3.2 scFv-based BsAbs
Single-chain variable fragments (scFvs) are the smallest antibody fragments that retain the antigen-binding specificity of the variable regions. ScFv-based BsAbs are typically produced by linking two scFvs together with a flexible linker. These BsAbs are smaller in size and have better tumor penetration than IgG-like BsAbs. However, they also have shorter half-lives and weaker effector function. Blinatumomab, the first BsAb approved for cancer therapy, is an example of an scFv-based BsAb that targets CD19 and CD3. scFv based BsAbs are known to rapidly clear from circulation due to their small size, which typically means they are administered by continuous infusion.
3.3 Fab-based BsAbs
Fab fragments consist of the variable and constant domains of the heavy and light chains. Fab-based BsAbs can be produced by chemically conjugating two Fab fragments together or by using recombinant DNA technology to express a single polypeptide chain containing two Fab fragments. These BsAbs have intermediate size and half-life compared to IgG-like and scFv-based BsAbs. Fab-based BsAbs can be engineered to have improved effector function by incorporating an Fc region or by recruiting effector cells through other mechanisms.
3.4 Tandem scFvs
Tandem scFvs or diabodies consist of two scFvs linked together with a short linker. The short linker forces the scFvs to dimerize, resulting in a bispecific molecule. Diabodies have better tumor penetration than IgG-like BsAbs but shorter half-lives. Triabodies and tetrabodies are similar to diabodies but contain three or four scFvs, respectively. These formats can have increased avidity and improved tumor targeting.
3.5 Dual-Affinity Re-Targeting (DART) Antibodies
DART antibodies are a type of scFv-based BsAb that contains two scFvs linked together by disulfide bonds. The disulfide bonds stabilize the molecule and improve its half-life. DART antibodies have been shown to be effective in redirecting T cells to kill tumor cells.
The choice of BsAb format depends on the specific application and the desired properties of the molecule. IgG-like BsAbs are generally preferred for applications that require long half-lives and strong effector function. scFv-based BsAbs are preferred for applications that require good tumor penetration. Fab-based BsAbs offer a balance between size, half-life, and effector function. In addition to the format, other factors such as the affinity of the binding domains, the linker length, and the glycosylation pattern can also affect the properties of the BsAb.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
4. Clinical Applications in Cancer
BsAbs have shown promising clinical activity in a variety of cancer types. Some of the most promising applications include:
4.1 Hematological Malignancies
BsAbs have been particularly successful in treating hematological malignancies, such as lymphomas and leukemias. Blinatumomab, which targets CD19 and CD3, has been approved for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). Mosunetuzumab, epcoritamab, and glofitamab, which target CD20 and CD3, have been approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (DLBCL). These BsAbs redirect T cells to kill lymphoma cells, resulting in high response rates and durable remissions. BsAbs targeting other hematological malignancies, such as multiple myeloma and Hodgkin lymphoma, are also under development. A key advantage of BsAb therapy for hematologic malignancies is that the target antigens are typically highly expressed and relatively specific to the malignant cells, reducing the risk of off-target toxicity. Furthermore, the accessibility of hematologic malignancies to circulating immune cells makes them particularly amenable to T-cell redirection strategies.
4.2 Solid Tumors
The clinical development of BsAbs for solid tumors has been more challenging due to the complex tumor microenvironment and the difficulty of achieving sufficient tumor penetration. However, several BsAbs targeting solid tumors are currently in clinical trials. One approach is to target tumor-associated antigens that are highly expressed on solid tumors, such as EGFR, HER2, and CEA. BsAbs targeting these antigens can induce receptor internalization, block signaling pathways, or deliver cytotoxic payloads to tumor cells. Another approach is to target the tumor microenvironment, such as the vasculature or the immune cells that infiltrate the tumor. BsAbs targeting VEGF or PD-L1 can inhibit angiogenesis or enhance anti-tumor immunity, respectively. A major hurdle for solid tumor BsAbs is overcoming the physical barriers that prevent adequate drug penetration. Strategies to improve tumor penetration include using smaller BsAb formats, modifying the glycosylation pattern, or combining BsAbs with other therapies, such as chemotherapy or radiation therapy. Another significant challenge is the heterogeneity of solid tumors, which can lead to resistance to BsAb therapy. Addressing this heterogeneity may require the development of multi-specific antibodies that target multiple antigens or signaling pathways.
4.3 Overcoming Resistance Mechanisms
Resistance to BsAb therapy can develop through various mechanisms, including antigen loss, downregulation of target receptors, and immune evasion. BsAbs can be designed to overcome these resistance mechanisms. For example, a BsAb targeting two different epitopes on the same antigen can prevent antigen loss from leading to resistance. Similarly, a BsAb targeting both the target antigen and an immune checkpoint can overcome immune evasion. Another approach is to combine BsAbs with other therapies, such as chemotherapy, radiation therapy, or other immunotherapeutic agents. Combinatorial strategies can enhance the efficacy of BsAbs and prevent the development of resistance.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
5. Safety Considerations
BsAb therapy can be associated with various adverse events, including:
5.1 Cytokine Release Syndrome (CRS)
CRS is a systemic inflammatory response that can occur when T cells are activated and release large amounts of cytokines. CRS can range from mild flu-like symptoms to life-threatening organ failure. The risk of CRS is particularly high with CD3-engaging BsAbs that potently activate T cells. Strategies to mitigate CRS include step-up dosing, pre-treatment with corticosteroids, and the use of tocilizumab, an anti-IL-6 receptor antibody. The development of lower-affinity CD3-binding BsAbs has also helped to reduce the incidence and severity of CRS. Predictive biomarkers for CRS are under investigation to identify patients who are at higher risk of developing this complication.
5.2 Immune-Related Adverse Events (irAEs)
BsAbs that target immune checkpoints can cause irAEs, which are autoimmune-like reactions that can affect any organ system. The management of irAEs typically involves corticosteroids and other immunosuppressive agents. Severe irAEs may require discontinuation of BsAb therapy. Similar to CRS, predictive biomarkers for irAEs are being investigated to personalize treatment and minimize toxicity.
5.3 On-Target, Off-Tumor Toxicity
BsAbs can also cause on-target, off-tumor toxicity if the target antigen is expressed on normal tissues. This type of toxicity can be minimized by selecting target antigens that are highly specific to tumor cells and by using BsAb formats with lower affinity for the target antigen. Careful monitoring of patients for signs of on-target, off-tumor toxicity is essential during clinical trials.
5.4 Infusion-Related Reactions (IRRs)
IRRs are acute reactions that can occur during or shortly after the infusion of a BsAb. IRRs can range from mild skin rashes to severe anaphylaxis. The management of IRRs typically involves stopping the infusion and administering antihistamines or corticosteroids. Pre-medication with antihistamines and corticosteroids can help to prevent IRRs.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
6. Future Directions
The field of BsAb therapy is rapidly evolving, with several promising future directions:
6.1 Multi-Specific Antibodies
Multi-specific antibodies can bind to more than two targets simultaneously, offering the potential to further enhance anti-tumor activity. For example, a tri-specific antibody could target a tumor-associated antigen, a T-cell surface marker, and an immune checkpoint. Multi-specific antibodies are more complex to design and manufacture than BsAbs, but they have the potential to overcome resistance mechanisms and enhance anti-tumor immunity.
6.2 Novel Target Combinations
Identifying novel target combinations is critical for expanding the clinical applications of BsAbs. For example, combining a T-cell redirecting BsAb with an immune checkpoint inhibitor may enhance anti-tumor immunity. Other promising target combinations include targeting multiple signaling pathways or targeting the tumor microenvironment.
6.3 Enhanced Delivery Strategies
Improving the delivery of BsAbs to the tumor microenvironment is essential for maximizing their efficacy in solid tumors. Strategies to enhance delivery include using smaller BsAb formats, modifying the glycosylation pattern, and combining BsAbs with other therapies, such as chemotherapy or radiation therapy. The use of nanoparticle-based delivery systems can also improve the tumor accumulation of BsAbs.
6.4 Conditional Activation
Developing BsAbs that are conditionally activated in the tumor microenvironment can improve their safety profile. For example, a BsAb that is activated by proteases that are specifically expressed in the tumor microenvironment would minimize off-target toxicity. Conditional activation can be achieved through various mechanisms, such as masking the binding domains with peptides that are cleaved by tumor-associated proteases.
6.5 Next-Generation BsAb Formats
New BsAb formats are being developed to improve their stability, effector function, and pharmacokinetic properties. For example, BsAbs with increased Fc region binding to FcγRs can enhance ADCC. BsAbs with improved half-life can reduce the frequency of dosing. BsAbs with reduced immunogenicity can minimize the risk of anti-drug antibodies.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
7. Conclusion
Bispecific antibodies represent a promising class of immunotherapeutic agents with the potential to revolutionize cancer therapy. Their ability to simultaneously engage two distinct targets allows for a broad spectrum of therapeutic possibilities, including T-cell redirection, immune checkpoint blockade, receptor clustering, and delivery of therapeutic payloads. The clinical development of BsAbs has been particularly successful in hematological malignancies, and ongoing clinical trials are exploring their potential in solid tumors. While BsAb therapy can be associated with various adverse events, such as CRS and irAEs, strategies to mitigate these toxicities are being developed. The field of BsAb therapy is rapidly evolving, with several promising future directions, including multi-specific antibodies, novel target combinations, enhanced delivery strategies, and conditional activation. BsAbs are poised to become a cornerstone of cancer immunotherapy in the years to come. In summary, this review has highlighted the versatility of bispecific antibody technology, and we expect to see an increasingly broad range of applications of this approach in the near future.
Many thanks to our sponsor Esdebe who helped us prepare this research report.
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