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Bispecific antibodies (BsAbs) are redefining the landscape of therapeutic design, offering dual-target precision that conventional antibodies can’t match. These specially engineered antibodies bind to either two different antigens or two distinct sites on the same antigen. BsAbs form a powerful new class of therapy which has caused excitement among the drug development community due to its flexibility, precision, and potential to treat conditions such as cancer.
The emergence of BsAbs represents a broader shift toward tailored, powerful, precision medicine, where treatments harness the body’s immune system or attack a disease through multiple targets. Developing this type of drug requires comprehensive knowledge of their mechanisms of action, pharmacokinetic (PK) characteristics, and the best strategies to ensure a smooth path to market.
Bispecific antibodies’ mechanisms of action
Many BsAbs are engineered to retain the Fc region, a part of the antibody that enables immune functions such as prolonged serum half-life, antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Other BsAbs, engineered without the Fc domain, have shorter half-lives, smaller molecular weights, enhanced tumor tissue penetration, and reduced immunogenicity.
To date, about 20 BsAbs have been approved globally: most of them target cancers, and three are used to treat hemophilia, ophthalmic diseases, and psoriasis. These therapies have four primary mechanisms of action.
- Bispecific T cell engagers. BsAb binds the CD3 subunit of the T cell receptor (TCR) and the selected tumor-associated antigen (TAA) to facilitate the formation of a synapse between the two cell types, which activates T cells, releasing perforins and granzymes to lyse tumor cells.
- Immune checkpoint (ICP) modulation. This includes dual ICP-blocking BsAbs and BsAbs targeting ICP concurrently with a target involved in other signaling pathways.
- Signaling pathway blockade. (i) BsAbs simultaneously bind two targets involved in signaling pathways. (ii) Biparatopic bsAbs bind to two separate epitopes on the same target.
- Functional mimicry. BsAbs are designed as a cofactor mimetic for the precise positioning of a substrate.
Dose linearity and prediction
BsAbs, like mAbs, often exhibit nonlinear pharmacokinetics at lower doses due to target-mediated drug disposition (TMDD). During this process, the drug binds to its target so strongly that it affects absorption, distribution, and clearance.
For example, amivantamab, which targets EGFR/c-Met, shows linear pharmacokinetics at high doses in testing, but at lower doses, the drug’s behavior becomes less predictable, likely due to TMDD.
When designing human trials of BsAbs, sponsors must exercise caution when predicting the first in human (FIH) dosage. TGN1412, a CD28-targeted T-cell agonist, taught researchers an important lesson. High CD3 affinity in BsAbs may overactivate T cells, potentially triggering dangerous cytokine storms. If the CD-3 binding strength is too high or the dose is too large, this can lead to serious side effects.
To ensure safety, the Minimum Anticipated Biological Effect Level (MABEL) approach is recommended to select a very conservative starting dose in early human trials.
Establishing ADME for bsAbs
Absorption: BsAbs have poor stability in the gastrointestinal tract and suffer low permeability across the gut wall, meaning they have negligible oral bioavailability. Most are administered via intravenous (IV), subcutaneous (SC), or intramuscular (IM) injection. In 2023, an orally administered BsAb SOR102 entered the clinic stage, composed of two nanobodies with a linker, targeting both TNF-α and IL-23p19. It showed very good stability in the gastrointestinal tract, aiming to deal with illness in the intestines.
Distribution: None of the marketed BsAbs conducted distribution studies. Similar to monoclonal antibodies, they remain in the circulatory system and extracellular fluids due to their large molecular size. This limits entry into cells and deep tissues.
Metabolism and Excretion: Like monoclonal antibodies, BsAbs are primarily cleared through proteolytic catabolism into short peptides or amino acids by cells of the reticuloendothelial system, including liver and endothelial cells, which then re-enter the body’s amino acid cycle or excrete through the kidney. Due to their protein-based nature, traditional metabolic enzyme pathways (e.g., CYP450) are not involved. As a result, metabolism and excretion are not typically a major concern in ADME assessments of BsAbs, and dedicated elimination studies are often limited or unnecessary for regulatory submission.
Bioanalysis of bispecific antibodies
Researchers primarily use ligand-binding assay platforms for the bioanalysis of BsAbs. Due to their structural complexity, four detection formats are employed: (i) detection of total antibody, (ii) detection of target 1, (iii) detection of target 2, and (iv) detection of intact antibody. Ligands serve as capture or detection reagents, and anti-idiotype antibodies can also be used.
In the “Bispecific Antibody Development Programs: Guidance for Industry” issued by FDA in 2021, it is mentioned that bispecific antibodies may present as a mixture of biologically active (e.g., unbound and capable of binding a ligand) and inactive forms (e.g., bound and not capable of binding a ligand) in
biological matrices. Consideration should be given to identifying the bispecific antibody forms most relevant to pharmacokinetic/pharmacodynamic assessment and to developing validated assays to measure the appropriate forms accordingly. Therefore, more than one assay may be recommended to quantify appropriate forms.
Researchers should select the detection format and critical reagents based on target information, antibody structure, and the stage of research. U.S. regulators recommend using multiple detection methods to characterize the structural integrity and metabolic status of BsAbs jointly. It’s also advised to look out for interference or overlap of data between methods.
BsAbs, which are designed as probodies, may have masking peptides and produce multiple active forms after metabolism. Due to their increased complexity, ligand-binding assays may not be adequate, and liquid chromatography-mass spectrometry (LC-MS) is more suitable and can detect surrogate peptides.
Immunogenicity of bispecific antibodies
The artificial structure of BsAbs increases immunogenicity risks, reduces safety and efficacy, and can lead to the formation of anti-drug antibodies (ADA), inducing serious drug-related toxicity. However, clinical data from approved T-cell engagers indicate low rates of ADA. For example, in clinical studies of emicizumab, only 2.8% of patients developed ADAs, and in glofitamab clinical studies, only 1.1% developed ADAs. No ADAs were detected in clinical studies for mosunetuzumab, possibly due to its mechanism of action, which activates T cells to kill B cells, leading to B cell depletion. However, immunogenicity poses an emerging challenge in the development of bispecific antibodies targeting solid tumors. Many BsAb-based immunotherapies for cancer discontinued further clinical development due to the formation of ADA.
Total ADA analysis usually employs the classic bridging assay format, using a three-step approach for screening, confirmation, and titer analysis.
Immunogenicity studies for BsAbs should be tailored to the specific research phase in which they are conducted. In early preclinical studies, analyzing total ADAs can aid in interpreting PK and TK data. During the IND stage, researchers should include analyses of immunotoxicity and immune cell profiling. In the clinical research phases, further assessments of immunotoxicity or neutralizing antibodies (Nabs) may be necessary.
Mitigating the risk of cytokine release syndrome
As previously mentioned, BsAbs’ high affinity for CD3 can lead to T-cell activation even before they bind to tumor cells, triggering the rapid and severe release of cytokines, which may result in a cytokine storm.
To mitigate this risk, sponsors can consider using pretreatments such as Obinutuzumab before administration. Clinical recommendations for stepwise dose escalation can also reduce toxicity and reduce the risk of cytokine storm. However, due to the potential severity of side effects, some drugs have been released a boxed warning.
In June 2023, U.S. regulators issued guidance on drug interactions for therapeutic proteins, explaining that some drugs that promote the release of cytokines, such as blinatumomab, may increase the level of pro-inflammatory cytokines and down-regulate the expression of cytochrome P450 (CYP450) enzyme, reducing the metabolism of drugs as substrates of CYP450 and increasing their exposure level.
Because most marketed BsAbs carry a risk of cytokine release, their use alongside CYP450 substrates may require monitoring and dose adjustments.
A final word
BsAbs offer enhanced specificity and reduced off-target toxicity by simultaneously targeting two antigens. This makes them a promising frontier in the development of antibody drugs. There are still challenges to overcome, as highlighted by the BsAbs already on the market, including managing the risk of cytokine storms, monitoring drug interactions, neurotoxicity, and more.
Bispecific antibodies are rapidly transforming therapeutic strategies, particularly in oncology and immunology, by enabling dual-target precision with reduced off-target effects. Yet their complexity demands equally sophisticated development strategies — especially in pharmacokinetics, bioanalysis, and immunogenicity assessment. As emerging formats like trispecifics and bispecific ADCs gain traction, the path from discovery to clinic will increasingly rely on integrated, modality-specific expertise. Forward-looking sponsors will benefit from engaging partners with deep, cross-functional capabilities in biologics development to ensure each therapeutic candidate is rigorously characterized, safe, and ready for regulatory advancement.
Dr. Qigan Cheng is currently a senior Study Director in the DMPK Service Department of WuXi AppTec. He has over 10 years of research experience in drug discovery and development and 6 years of research experience in preclinical pharmacokinetics. He has successfully supported over 30 new drug IND applications globally.
Filed Under: Biologics
