Bispecific antibodies represent a major innovation in therapeutic development, offering the unique ability to bind two distinct targets simultaneously. This dual-targeting approach has opened new avenues for treating complex diseases, particularly in oncology and immunology.
Join us as we explore the world of bsAbs, discovering their formats, mechanisms of action, and therapeutic applications. Plus, find out how evitria’s bispecific antibody expression service is propelling biomedical research forward. Ready to embark on a journey into the realm of bispecific antibodies? Let’s travel together and unlock their full potential!
What is a bispecific antibody?
A bispecific monoclonal antibody (BsMAb) or bispecific antibody (BsAb) is an engineered protein designed to bind simultaneously to two different antigens or epitopes. This dual-targeting ability surpasses the limitations of naturally occurring monoclonal antibodies, unlocking expanded therapeutic potential and offering new possibilities for disease treatment.
BsAbs are engineered in various formats, including IgG-like structures and single-chain constructs, each offering specific advantages in modulating immune responses and engaging target receptors. These formats enable a diverse range of mechanisms of action, activating the host immune system, carrying cytotoxic payloads, engaging T cells, and more.1
Beyond cancer immunotherapy, bsAbs have garnered significant interest in diverse fields such as drug delivery (through bispecific antibody-drug conjugates)2 and neurodegenerative diseases like Alzheimer’s.3 Their potential to target complex biological pathways and bridge multiple cell types positions them as promising candidates for addressing unmet medical needs across various therapeutic domains.
What is the difference between a monoclonal and a bispecific antibody?
The primary difference between monoclonal antibodies (mAbs) and bispecific antibodies is in their ability to target antigens. Monoclonal antibodies are designed to bind to a single, specific antigen or epitope. This makes them highly effective in targeting and neutralizing one particular molecule or pathogen, which has been the basis for many successful therapies.
Bispecific antibodies, on the other hand, have the unique ability to bind to two different antigens or epitopes simultaneously. This dual-targeting capability enables bsAbs to perform more complex functions that are not possible with monoclonal antibodies alone. For instance, a bsAb might bind to a cancer cell with one arm and a T cell with the other, bringing the immune system directly into contact with the tumor to enhance its destruction.
This versatility opens up expanded therapeutic applications, making bispecific antibodies a promising approach for treating multifaceted diseases like cancer, infectious diseases, and autoimmune disorders, where multiple biological pathways or targets may need to be addressed simultaneously.
Historical background of bispecifics
BsAbs were first discussed in the early 1960s,4 receiving major attention in the decade to come. However, their reliable production remained a challenge. In 1983, quadromas were described by Milstein.5 These cell lines (also known as hybrid hybridomas) were able to produce bispecifics, but were far from perfect, as the frequency of mispairing between antibody fragments was rather large.
Over subsequent decades, advancements in recombinant DNA technology and protein engineering propelled the evolution of bsAbs. These developments paved the way for novel formats with enhanced specificity and functionality, laying the foundation for therapeutic applications.
In 2009, the approval of catumaxomab (Removab®) by the European Medicines Agency for the treatment of malignant ascites in EpCAM-positive carcinomas6 represented the first bsAb to see regulatory approval and marked a significant milestone in bsAb history. This was followed by the FDA approval of blinatumomab (Blincyto®) in 2014 for relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL), further validating the therapeutic potential of bsAbs.7
Today, bsAbs represent promising candidates for treating various diseases, including cancer, autoimmune disorders, and infectious diseases. The historical journey of bsAbs underscores the ongoing pursuit of innovation in biomedical research and therapeutic interventions.8
Bispecific antibody formats
Bispecific antibodies encompass a diverse array of formats and engineering strategies, each tailored to optimize therapeutic efficacy and target engagement.
1. IgG-like Structures
IgG-like bispecific antibodies mimic the structure of natural immunoglobulins, comprising two antigen-binding fragments (Fab) fused to the constant regions of IgG molecules. This format offers stability, prolonged circulation, and effector functions, making it suitable for systemic administration and immune cell recruitment.
2. Non-IgG-like Structures
Non-IgG-like bsAbs are engineered fusion proteins, lacking the stable Fc region. These constructs leverage alternative binding domains such as individual single-chain variable fragments (scFvs), Fab domains, or VHH domains, offering unprecedented flexibility and modularity in target engagement. This smaller format facilitates compact design, enhanced tissue penetration, and efficient production, enabling diverse therapeutic applications.
Beyond structural considerations, bsAbs can be classified based on their valency, reflecting the number of binding domains present and their distribution among different targets. Common valencies include “1 + 1” (two binding sites, one for each target), “2 + 2” (four binding sites, two for each target), and “2+1” (three binding sites, two for one target, one for the other). These various arrangements allow for engagement of multiple targets with varied affinity, and can be optimized for a specific application.
Antibody engineering and characterization
The development of bispecific antibodies relies on sophisticated engineering techniques to precisely manipulate antibody structure and function. Central to this process is the strategic incorporation of antigen binding sites, ensuring optimal target recognition and immune cell activation. Techniques such as knobs-into-holes mutagenesis and peptide linker optimization enable the generation of bsAbs with improved efficiency, tailored properties and functionalities.
Assessing the efficacy and specificity of bispecific antibodies requires robust characterization methods to validate their therapeutic potential. Advanced analytical techniques, including surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISA), enable quantitative assessment of binding affinity and target specificity. Additionally, functional assays, such as cytotoxicity assays and receptor blockade assays, provide insights into bsAbs’ ability to induce immune cell activation and target engagement.
At evitria, we offer extensive support for researchers exploring the world of bsAbs. With over 25,000 antibodies and antibody-related molecules produced using our transient CHO cell expression system, we possess unparalleled expertise in bsAb expression. Our team can guide you through the selection of optimal formats, whether it’s the widely-used Duobody system from Genmab9 or innovative formats like IgG-scFvs. With our support, researchers can navigate the complexities of bsAb research and accelerate their path towards groundbreaking discoveries.
Mechanisms of action
Dual Binding
BsAbs possess the unique capability to simultaneously target antigens present on cancer cells and T cells. By binding to both targets, bsAbs facilitate the formation of immunological synapses between cancer cells and cytotoxic T cells, triggering a cascade of immune responses culminating in cancer cell lysis.
Effector Cell Activation and Cytokine Release
Antibody-dependent cellular cytotoxicity (ADCC) is central to the immune response to cancer cells. In this process, antibodies bound to antigens on the surface of cancerous cells recruit and activate effector immune cells, such as natural killer cells, for tumor recognition and elimination. Upon engagement with bsAbs, effector cells undergo robust activation, releasing cytotoxic molecules and pro-inflammatory cytokines into the tumor microenvironment.
Dynamic Interactions with Target Receptors
By engaging with target receptors expressed on cancer cells, bsAbs modulate signaling cascades that control various cellular functions, including proliferation, survival, and apoptosis. The binding between bsAbs and target receptors elicits changes in downstream signaling, driving anti-tumor immune responses and inhibiting cancer cell growth and metastasis.
Therapeutic Applications of Bispecific Antibodies
Bispecific antibodies have demonstrated promising applications across a spectrum of therapeutic domains, revolutionizing treatment paradigms beyond cancer therapy.
Hematologic Malignancies
In hematologic malignancies such as acute lymphoblastic leukemia (ALL) and multiple myeloma, bispecific antibodies have emerged as transformative therapies, offering new hope for patients with refractory and relapsed cancers. Clinical trials and FDA-approved therapies showcase the efficacy and safety profile of bsAbs in targeting malignant cells while sparing healthy tissues.10 With their potential to induce potent anti-tumor activity in hematologic malignancies, bsAbs represent a promising avenue for improving patient outcomes in conditions like B-cell lymphoma.11
Solid Tumors
Ongoing research and clinical development efforts focus on harnessing the potential of bsAbs for solid tumors, including lung cancer, breast cancer, and colorectal cancer. Despite challenges in on-target off-tumor toxicity and high necessary doses,12 bsAbs hold promise as targeted therapeutics, offering opportunities to overcome treatment resistance and enhance patient outcomes. BsAbs may one day pave the way for personalized and precision medicine approaches in tumor oncology, particularly in HER2-positive breast cancer where they can unleash potent anti-tumor activity.13
Further Therapeutic Applications
Beyond the realm of oncology, bispecific antibodies demonstrate versatility in addressing diverse therapeutic needs across a range of diseases. From autoimmune disorders to infectious diseases, bsAbs offer targeted interventions with the potential to modulate immune responses and restore homeostasis.14
Whether as modulators of immune checkpoints or as targeted inhibitors of pathogenic molecules, bsAbs represent a transformative class of therapeutics poised to redefine the treatment landscape across multiple therapeutic areas.
Advantages of bispecific antibodies
The numerous advantages of bispecific monoclonal antibodies make them fit for a large variety of uses. These benefits mainly derive from the innovative structure and mechanism of bsAbs, as they are able to bind to multiple targets at the same time.
This makes them extremely effective and versatile, e.g. in cancer therapy. Furthermore, they can reduce side effects due to their high specificity and targeted mechanism.
Potential side effects in bsAb therapy
Despite being extremely promising therapeutic means, there are also side effects related to their administration. These can be grouped into infusion-related, immune-related, hematologic, and organ specific side effects.
The occurrence of side effects in bsAb therapy depends on patient-specific factors, the exact bispecific antibody and its administration, as well as its target. In order to avoid side effects respectively soften their impact, it is necessary to individually evaluate if a patient may receive a bsAb treatment.
Once selected, the treatment must be adjusted to the patient’s prerequisites and reactions, and the symptoms of potential side effects must be treated in order to improve the patient’s comfort.15
FDA Approved Bispecific Antibodies
The FDA has granted approval to several bsAb therapies, marking significant milestones in cancer treatment.
Early Pioneer: Blinatumomab (Blincyto®, Approved in 2014)
Blinatumomab, an early example of FDA-approved bispecific antibodies, revolutionized the treatment landscape for relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). By engaging CD19 on B cells and CD3 (anti-cd3) on T cells, blinatumomab induces targeted cytotoxicity, leading to remission and improved survival outcomes in patients with limited therapeutic options.16
Recent Advancement: Tarlatamab (Imdelltra®, Approved in 2024)
Tarlatamab represents the latest addition to the arsenal of FDA-approved bsAbs, demonstrating efficacy in extensive stage small cell lung cancer (SCLC). Targeting DLL3 on SCLC cells and CD3 on T cells, tarlatamab mediates immune cell recruitment and tumor cell lysis, offering new hope for patients with this aggressive form of lung cancer.17
Future Prospects
The approval of bsAbs like blinatumomab and tarlatamab underscores the transformative potential of this innovative class of therapeutics. As research progresses, efforts are underway to enhance bispecific antibody therapies, focusing on minimizing off-target toxicity while maximizing anti-tumor activity, thereby improving patient outcomes.11
Here are approved bispecific antibodies listed by the FDA:
| Trade Name | Active Ingredient | Year Approved | Indication |
| Blincyto | blinatumomab | 2014 | To treat Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia |
| Hemlibra | emicizumab-kxwh | 2017 | To prevent or reduce the frequency of bleeding episodes in hemophilia A with factor VIII inhibitors |
| Rybrevant | amivantamab-vmjw | 2021 | To treat locally advanced or metastatic non-small cell lung cancer with certain mutations |
| Kimmtrak | tebentafusp-tebn | 2022 | To treat a form of unresectable or metastatic uveal melanoma |
| Vabysmo | faricimab-svoa | 2022 | To treat neovascular (wet) age-related macular degenerated and diabetic macular edema |
| Tecvayli | teclistamab-cqyv | 2022 | To treat relapsed or refractory multiple myeloma |
| Lunsumio | mosunetuzumab-axgb | 2022 | To treat relapsed or refractory follicular lymphoma |
| Epkinly | epcoritamab-bysp | 2023 | To treat relapsed or refractory diffuse large B-cell lymphoma |
| Columvi | glofitamab-gxbm | 2023 | To treat relapsed or refractory diffuse large B-cell lymphoma or large B-cell lymphoma |
| Ziihera | zanidatamab | 2024 | To treat HER2-expressing biliary tract cancers. A biparatopic IgG1 antibody developed by Zymeworks and Jazz Pharmaceuticals that targets two distinct epitopes on HER2. |
| Talvey | talquetamab | 2023 | To treat relapsed or refractory multiple myeloma. A bispecific DuoBody® antibody developed by Janssen Biotech that targets GPRC5D and CD3. |
| Elrexfio | elranatamab | 2023 | To treat relapsed or refractory multiple myeloma. A tandem scFv bispecific antibody developed by Pfizer that targets BCMA and CD3. |
| Imdellra | tarlatamab | 2024 | To treat small cell lung cancer (SCLC). A bispecific T-cell engager (BiTE®) developed by Amgen that targets DLL3 and CD3. |
| Bizengri | zenocutuzumab | 2024 | To treat NRG1 fusion–positive solid tumors, including NSCLC and pancreatic cancer. A bispecific antibody developed by Merus that targets HER2 and HER3. |
| Lynozyfic | linvoseltamab | 2025 | To treat relapsed or refractory multiple myeloma. A bispecific tandem scFv antibody developed by Regeneron that targets BCMA and CD3. |
Production of bispecific antibodies
The production of bsAbs is a multifaceted process that combines protein engineering with advanced manufacturing techniques. Here’s an overview of the key steps involved:
Antibody Engineering and Cloning
BsAbs are created by merging two distinct monoclonal antibodies (mAbs) with desired specificities. Using recombinant DNA technology, genes of each antibody are cloned to generate a single construct, combined in various ways to create diverse bispecific formats like single-chain variable fragments (scFv) or diabodies.
Transfection and Expression
Bispecific antibodies are synthesized using various expression systems, including mammalian cells, yeast, or bacterial systems. Mammalian cell lines such as CHO cells are preferred for therapeutic applications due to their capacity for complex post-translational modifications essential for antibody functionality. Following transfection, where the genes encoding the bsAb are introduced into the chosen expression system, the system initiates the production of bsAb molecules.
For quadroma expression systems, hybrid hybridomas are formed by fusing two distinct hybridomas producing the parent monoclonal antibodies. Chains from each monoclonal antibody then combine to create the bsAb, although erroneous homodimerization and heavy-light chain mismatches are common. Recombinant techniques like knobs-into-holes mutations can facilitate proper heavy chain heterodimerization, yielding asymmetric bsAbs.
Protein Purification
Bispecific antibody production necessitates multiple purification steps to obtain highly pure and active molecules. Techniques such as ion exchange chromatography, protein A affinity chromatography, or peptide tagging are utilized to isolate and enrich bsAbs from the expression system.
Antibody Characterization and Quality Control
Following purification, bsAbs undergo rigorous characterization and quality control assays to assess their binding specificity, stability, and effector functions. These assays also evaluate pharmacokinetics, half-life, and target cell engagement, ensuring consistent product quality.
Bispecific Antibody Expression Service from evitria
At evitria, we specialize in bispecific antibody production, offering comprehensive bispecific antibody expression services to meet the diverse needs of our clients. Leveraging Lonza’s bYlok® technology, we redefine IgG-like bispecific antibody design with simplicity and confidence, ensuring correct heavy-light chain (HC-LC) pairing.
Achieving accurate HC-LC pairing during bispecific antibody production is crucial, as it can impact yields and downstream processing efficiency. With bYlok® technology, bispecific antibodies generated at evitria have demonstrated over 95% correct HC-LC pairing, guaranteeing high-quality molecules for therapeutic development.
Our collaboration with Lonza enables us to provide early-stage bispecific antibody discovery customers with rapid transient production of variants for discovery and non-GMP development studies.
At evitria, we are committed to delivering cutting-edge bispecific antibody solutions, driving innovation in biopharmaceutical research and development. Find out more about all our recombinant antibody expression services.
Read more about Bispecific Antibodies
- Bispecific antibody production – A comprehensive overview
- Understanding the side effects of bispecific antibodies
- Development of bispecific antibodies: challenges & solutions
- How to choose a bispecific antibody format?
- Bispecific antibody formats
- What companies are developing bispecifc antibodies?
- FDA approved bispecifc antibodies
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