How to select the optimal bispecific antibody format for therapeutic success

Bispecific antibodies (bsAbs) have transformed the biologics landscape, offering the unique ability to simultaneously engage two different antigens or epitopes.¹ From redirecting immune cells to tumours, to dual-pathway inhibition or receptor clustering, bsAbs are driving new therapeutic strategies across oncology, immunology and beyond.²

Yet despite their promise, bsAbs are not a one-size-fits-all solution. Choosing the right format is a strategic decision that influences everything from mechanism of action and developability to manufacturability and regulatory success.³ At evitria, we’ve seen firsthand how early format choices can shape the entire course of therapy development.

This article provides a high-level guide to help you align bsAb format selection with your therapy development goals, covering the most common bispecific antibody formats in use today along with their respective advantages, limitations and use cases.⁴ Further, you’ll learn more about the strategic considerations that can inform smarter, faster decision-making throughout development.

Making the best decision: why bsAb format matters

Each bispecific antibody format is a compromise between biology and manufacturability.⁵ Your ideal format should align with:

• your biological goal (eg, T-cell engagement, receptor crosslinking, dual inhibition)
• the pharmacokinetic profile needed for therapeutic effect
• production scalability and purity standards necessary for clinical development.

Selecting the wrong format can result in suboptimal efficacy, poor expression yields, stability issues or costly re-engineering down the line.⁶

Figure 1: Mechanism of action of bispecific antibodies in redirecting immune cells to target cancer cells. The illustration shows bispecific antibodies simultaneously binding to a tumour cell and an immune cell, facilitating close contact between the two. This dual binding enables immune cells – such as T cells – to recognise and attack tumour cells effectively.  Image source: evitria AG 

Strategic format selection: key criteria to consider

When selecting a bsAb format, it is essential to align structural design with therapeutic function. Key considerations include:

Mechanism of action

The choice of bispecific antibody format is fundamentally driven by its intended mechanism of action. For example, T-cell engagers often favour Fc-less formats such as BiTEs and DARTs due to their compact size and ability to efficiently bring T cells into proximity with tumour cells.⁷

In contrast, dual-pathway inhibitors or receptor-clustering therapeutics typically require Fc-containing formats to ensure molecular stability and prolonged systemic exposure.

Importantly, the format and valency of different domains must align with biological parameters such as the distance between target antigens, their relative expression levels and the binding affinities of the variable domains. Recent evidence shows that for T-cell engagers, achieving stronger binding affinity to the tumour antigen than to CD3 on T cells is desirable to optimise efficacy and safety profiles.⁸

This can be accomplished by reducing the affinity of the anti-CD3 binding arm, thereby minimising off-tumour T-cell activation and cytokine release, or by engineering multiple tumour-binding arms for a single anti-CD3 domain, as exemplified by glofitamab.⁸

Additionally, the use of antibody fragment-based formats (eg, tandem scFvs) may be determined not only by mechanism of action requirements but also by production considerations, such as expression system compatibility and purification strategy.⁸

Fc effector function

The Fc region of a bispecific antibody plays a pivotal role in defining its therapeutic mechanism and pharmacokinetic behaviour. Retaining the Fc domain offers several key advantages, including engagement of immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). These mechanisms are  critical for effectively targeting and eliminating tumour cells or pathogenic targets in vivo.⁹

Additionally, the Fc region interacts with the neonatal Fc receptor (FcRn), facilitating recycling and thereby prolonging the serum half-life of the antibody. This is a key consideration for therapeutics intended for chronic administration or when extended exposure is necessary to achieve therapeutic efficacy.¹⁰

However, in applications where Fc-mediated effector functions could pose safety risks, such as in T-cell engagers that could trigger off-tumour toxicity, Fc silencing technologies are used to abrogate these interactions while retaining structural stability.⁹

Common approaches include LALA mutations, STR modifications and aglycosylation – each designed to reduce or eliminate Fcγ receptor binding without compromising expression or folding.⁹

Pharmacokinetics

The pharmacokinetic profile required for your therapeutic application is a critical determinant of bsAb format choice. Treatments targeting chronic conditions benefit from formats with extended half-life to reduce dosing frequency and maintain therapeutic exposure. Fc-containing formats are particularly suited for these applications due to their interaction with the neonatal Fc receptor (FcRn), which enables recycling and prolonged circulation.¹⁰

In contrast, acute interventions or applications where shorter systemic exposure is preferable, such as certain T-cell engager therapies to minimise cytokine release, may favour Fc-less constructs with inherently shorter half-lives.¹¹ Balancing therapeutic duration with safety and efficacy requirements is essential when selecting the optimal format for your molecule.

Expression system

The choice of expression system is closely tied to format complexity and required post-translational modifications. Fc-containing bsAbs, which require proper glycosylation for structural stability and effector function, are typically produced in mammalian expression systems such as CHO cells.¹²

Simpler constructs, including Fc-less formats like tandem scFvs or certain fragment-based bispecifics, can often be produced in microbial hosts such as E. coli, enabling rapid production with reduced cost. However, these systems may lack the capacity for human-like glycosylation, potentially limiting their use for applications requiring Fc-mediated functions.¹³

Purification

Purification strategies must reflect the structural complexity of the chosen format. IgG-like bispecifics, which are prone to heavy–light chain mispairing, benefit from high-fidelity solutions such as bYlok^® technology, achieving over 95 percent correct chain assembly to maximise yield and purity.¹⁴

Non-Fc formats, such as tandem scFvs and BiTEs, avoid mispairing challenges altogether due to their single-chain architecture. However, they present unique purification considerations. For example, blinatumomab, a BiTE construct, uses a 6xHis affinity tag for purification – a method generally avoided in GMP settings due to potential immunogenicity or regulatory concerns.¹⁵ Alternative purification strategies may therefore be needed to align with manufacturing standards for clinical-grade material.

Regulatory precedent

Leveraging formats with established regulatory precedent can help streamline development timelines and reduce approval risk. Proven engineering strategies like Knobs-into-Holes (KiH) combined with CrossMab designs have been validated in multiple clinical programmes, offering greater confidence in manufacturability and safety profiles.¹⁶

However, developers must also consider intellectual property (IP) restrictions associated with certain proprietary platforms. Early assessment of licensing requirements is critical to ensure freedom to operate and avoid delays in later-stage development and commercialisation.

Format selection in action: real-world examples

The diversity of approved bispecific antibody (bsAb) formats reflects how format choice directly supports therapeutic intent, spanning half-life, effector function, manufacturability and safety profiles.

Bispecific antibodies currently in clinical trials employ a wide range of mechanisms of action.

Figure 2: Schematic of bispecific antibody formats currently in clinical trials, designed to engage immune effector cells for targeted cancer cell killing. Adapted from: Klein, Christian et al. (2024) The present and future of bispecific antibodies for cancer therapy. Nature reviews Drug Discovery, 23(4), 301-319. doi:10.1038/s41573-024-00896-6

These include:

• T-cell engagement, such as CD3 x TAA constructs, to redirect T cells for targeted tumour killing
• Dual checkpoint blockade (eg, PD1 x CTLA4) to enhance immune activation against tumours
• Dual inhibition of tumour-associated pathways, such as VEGF x TGFβ, modulating the tumour microenvironment
• Co-stimulatory receptor targeting to amplify immune responses
• Macrophage and NK cell engagement, broadening therapeutic potential beyond T cells.

These modes are exemplified in Figure 3 from Klein et al. (2024), which illustrates formats such as BiTEs, DARTs, CrossMabs and dual inhibitors within ongoing clinical programmes.

Figure 3: Approved bispecific antibodies for cancer therapy, categorised into T-cell engagers (a) and non-T-cell engagers (b). T-cell engagers, such as Blinatumomab and Mosunetuzumab, link T-cells to target cancer cells. Non-T-cell engagers, like Amivantamab, target different receptors on cancer cells without directly engaging T-cells. Adapted from: Klein, Christian et al. (2024) The present and future of bispecific antibodies for cancer therapy. Nature Reviews Drug Discovery, 23(4), 301–319. doi:10.1038/s41573-024-00896-6.

The following table summarises approved bsAbs, highlighting their format, targets and therapeutic applications:¹⁶

How evitria supports your format evaluation

With dozens of available bsAb formats, the real challenge lies in selecting the one that aligns with your biological mechanism, manufacturing constraints and development timeline. At evitria, we understand that format selection isn’t only about protein engineering, but about aligning science, strategy and execution.

We help therapeutic developers make informed decisions by:

• Rapidly producing and comparing multiple bsAb formats at small scale
• Providing expert guidance on pairing technologies, such as our licensed bYlok^® system, to maximise correct assembly
• Offering Fc engineering support (eg, STR modifications) for tailoring effector functions
• Ensuring that format decisions are compatible with downstream purification and scalability.

Whether you’re developing a next-gen T-cell engager or evaluating Fc-enhanced bsAbs for solid tumours, we provide the infrastructure and insight to accelerate your path forward.

References

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