Bispecific Antibodies: A Comprehensive Overview

• Entity Definition: Bispecific antibodies (bsAbs) are engineered biologics capable of simultaneous engagement of two distinct epitopes, overcoming the monospecific limitation of standard monoclonal antibodies.
• The Production Bottleneck: Primary hurdles include heavy chain heterodimerization, heavy-light chain mispairing, domain instability, and the resulting impact on yield and product homogeneity.
• The CHO Advantage: Utilizing transient expression in Chinese Hamster Ovary (CHO) cells from the earliest discovery phases completely mitigates translational risk when moving to clinical manufacturing.
• Solving the bottleneck: Transient CHO expression combined with scFv-stabilization technologies such as Opti-mAb® can rescue unstable bispecific formats (like IgG-scFvs), reduce aggregation, and improve yield and thermal stability early in development.

This article reviews the main formats, mechanisms of action, and therapeutic applications of bispecific antibodies, and outlines how they are produced in CHO cells. It also shows how evitria’s bispecific antibody production service supports researchers working with these molecules.

What is a bispecific antibody?

A bispecific monoclonal antibody or bispecific antibody 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 healthcare and 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 However, it historically introduced severe manufacturing bottlenecks, specifically heavy and light chain mispairing. Solving this biochemical hurdle early in the R&D phase is critical for downstream developability.

Unlike a monoclonal antibody, which binds a single target, a bispecific antibody binds two, which enables more complex mechanisms. For a full comparison, see our article on the difference between monoclonal and bispecific antibodies.

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. Random chain association in quadromas yields up to 10 different antibody species, with the desired bispecific representing only ∼12.5% of total product for standard IgG formats.

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. It was later voluntarily withdrawn in 2017 for commercial reasons. 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 bispecific antibody 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 canonical IgG architecture. 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. The strategies behind these molecules are covered in our piece on engineering bispecific antibodies.

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.

The choice of format, from the widely used DuoBody® system (from Genmab⁹) to IgG-scFvs, has consequences well beyond binding, which is why developability deserves a closer look.

Developability

Developability describes how easily a promising bispecific design can be turned into a stable, manufacturable product, and for bispecifics it is decided early. Three factors matter most.

1. First, early assessment of expression and stability shows whether a construct can be produced at useful yield before resources are committed.
2. Second, analytical characterization, for example by LC-MS and size exclusion chromatography, detects mispairing and aggregation that would otherwise surface only later.
3. Third, format selection sets the ceiling for what is achievable, since some formats are inherently easier to pair and purify than others.

Addressing these points during design and engineering, rather than after, is what separates a candidate that merely binds from one that can actually be made.

Mechanisms of action

Understanding how bispecific antibodies work requires looking at four primary functions: dual binding, immune effector cell recruitment, receptor modulation, and non-immune biochemical bridging.

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 immune effector 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.

Non-immune mechanisms

Not all bispecific antibodies act by recruiting immune cells. Some use their two arms for purely biochemical tasks. Emicizumab (Hemlibra) works as a cofactor mimetic, bridging activated Factor IX and Factor X to substitute for the missing Factor VIII in hemophilia A. Faricimab (Vabysmo) relies on dual pathway inhibition, blocking both VEGF-A and Ang-2 to reduce vascular leakage in retinal disease.

Bispecific designs are also being explored for viral neutralization, where one arm anchors the virus while the other blocks cell entry, an approach that can cover escape mutants better than a single antibody. Together, these examples show that the dual-targeting principle extends well beyond immune cell engagement.

Another non-immune application is drug delivery across the blood-brain barrier. In the transferrin-receptor brain shuttle approach, one arm binds the transferrin receptor on brain endothelial cells to ferry the antibody into the central nervous system, while the second arm engages the actual therapeutic target. This strategy is being explored for neurodegenerative diseases such as Alzheimer’s, where getting antibodies into the brain is a major hurdle.

Therapeutic Applications and approved products

Because bispecific antibodies bind two targets at once, they enable therapeutic strategies that single-target antibodies cannot. This makes them versatile across many diseases, and their targeted mechanism can improve efficacy while helping to limit off-target effects. These advantages are already visible across the clinical applications and approved products below.

Hematologic Malignancies

In hematologic malignancies (blood cancer) 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

Hemophilia A

Not every bispecific antibody engages the immune system. Emicizumab (Hemlibra) treats hemophilia A by acting as a functional substitute for missing Factor VIII. It bridges activated Factor IX and Factor X, bringing them into the correct orientation so that coagulation can proceed. This makes emicizumab a clear example of a bispecific used as a molecular tool rather than a targeting agent, extending the format well beyond oncology.

Ophthalmology

In ophthalmology, faricimab (Vabysmo) inhibits two drivers of vascular leakage at the same time, VEGF-A and Ang-2. By blocking both pathways with a single molecule, it treats retinal diseases such as neovascular age-related macular degeneration and diabetic macular edema, and can extend the interval between injections. Faricimab shows how the dual-targeting principle translates into chronic, non-oncologic disease.

Approved products

As of August 2026, 15 bispecific antibodies have received FDA approval, spanning formats from tandem scFv and BiTE to DuoBody and CrossMab, and indications from leukemia and lymphoma to solid tumors, hemophilia, and eye disease. For the complete, sourced list with targets and formats, see our overview of FDA-approved bispecific antibodies.

Further Therapeutic Applications

Beyond these approved examples, bispecific antibodies are being explored across a much wider range of conditions, including autoimmune and infectious diseases, and increasingly in neurology, where brain-penetrant bispecific designs are under investigation for disorders such as Alzheimer’s.

Whether as modulators of immune checkpoints or as targeted inhibitors of pathogenic molecules, they point to a therapeutic class that continues to expand well beyond its origins in oncology.

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

While early bispecific fragments (like scFvs) are sometimes evaluated in bacterial or yeast systems, this introduces severe translational risk. Different expression hosts yield radically different folding efficiencies, stability profiles, and glycosylation patterns.

To completely de-risk scale-up, bispecific antibodies must be synthesized in Chinese Hamster Ovary cells from day one of discovery. CHO platforms ensure native human-like post-translational modifications (PTMs) critical for stability and effector function (e.g., ADCC). By utilizing transient CHO expression during early discovery, large pharma and biotech developers eliminate the batch-to-batch variability and sequence-to-function discrepancies that cause late-stage clinical failures.

For quadroma systems or recombinant approaches utilizing knobs-into-holes mutations, expressing directly in CHO ensures that the heterodimerization efficiencies observed in discovery will directly translate to stable cell line development.

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.

Catching stability and expression problems early is critical, as we explain in what can go wrong in bispecific development.

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 bispecific antibody treatments 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.

The most notable immune-related adverse event are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). CRS occurs when the immune system responds too aggressively to the treatment, resulting in a “cytokine storm” that can cause low blood pressure, fever, nausea, and in severe cases, multi-organ failure. ICANS specifically affects the central nervous system. Symptoms of ICANS can range from mild confusion and tremors to more severe expressive aphasia or seizures.

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.¹⁴

Producing your bispecific antibody with evitria

As a CHO specialist, evitria supports researchers in producing complex bispecific formats by transient expression. Our proprietary Opti-mAb® technology stabilizes demanding scFv-based constructs, reducing aggregation and improving yield and thermal stability, so a promising design translates into stable, research-ready material.