Resources Blog Antibody Basics: Part 3​ - Antibody formats: Bispecific Antibodies (bsAbs)​

Antibody Basics: Part 3​ - Antibody formats: Bispecific Antibodies (bsAbs)​

Biointron 2024-02-14 Read time: 10 mins

Welcome back to Antibody Basics by Biointron, Part 3. In this episode, we’ll cover bispecific antibodies and other multispecific formats.

Engineered antibodies are modified versions of antibodies that have been designed to improve certain properties or functionalities, such as to enhance their therapeutic or diagnostic potential. 

  • Monoclonal antibodies (mAbs) have two identical binding sites which each recognize the same target antigen.

  • Bispecific antibodies (bsAbs) have two unique binding sites directed at two different antigens or two different epitopes on the same antigen.


  • 1960s: The first bispecific antibodies were described when Fabs from two polyclonal sera were re-associated into F(ab')2 molecules after reduction and reoxidation.

  • 1980s: The first monoclonal Abs were generated through hybridoma technology.

  • 1992: The first article demonstrating the therapeutic potential of bsAbs was published.

  • 2000s: The first approvals of bsAbs for therapeutic use (catumaxomab and blinatumomab) took place.

  • Present: Ongoing advancements in antibody engineering and further approvals of bsAbs.

Why choose bispecific antibodies?

Multispecific antibodies can bind to more than one target simultaneously, thus have higher binding specificity. They are advantageous over mAbs in clinical therapeutic effects, as improved function translates into improved response rates. In cancer, direct specific effectors of the immune system is used to target tumor cells, enhancing cytotoxicity. For optimization of cost, it is an alternative to combination therapy with two monospecific drugs.


Image credit: DOI: 10.3389/fimmu.2021.626616

Choosing an appropriate format

1. Single-chain-based format

  • Small size allows them to be used to link immune cells to tumor cells, creating an immunological synapse

  • Rapidly cleared from the blood within minutes

  • Better diffusion into the tumor tissue

  • Higher potency due to closer proximity of interactions in the two paratopes

  • Ease of large-scale production in microbial systems

  • Less immune-related adverse effects due to lack of Fc

2. IgG-based format

  • Extended blood half-life (~a few weeks), due to recycling by neonatal Fc receptor (FcRn)

  • Possess own crystalline fragment (Fc)-mediated functions

  • Larger than single-chain-based format 

  • Longer glomerular preservation 

  • Improved solubility and stability

  • Triggers cytotoxic and T-cell priming effects 

  • Can be purified by affinity chromatography workflows 

Popular bsAb formats

Knobs-into-holes: Replace a smaller amino acid with a larger amino acid in the CH3 region of an antibody chain to form “knobs”. Meanwhile, replace a larger amino acid in the other chain with a smaller amino acid to form “holes”.

Dual-Variable-Domain-IgG (DVD-Ig): A symmetrical molecular platform with four antigen binding sites and one pair of Fab domains. Each antibody arm utilizes short peptides to connect two variable regions.

Bispecific T-cell engager (BiTE): Link the CD3 antibody scFv and the tumor-associated antigen to redirect T cells to cancer cells for more targeted killing.

Bispecific antibodies are particularly useful as therapeutics in cancer

  • Redirect immune effector cells to the proximity of tumor cells

  • Block different signaling pathways simultaneously

  • Target tumor angiogenesis 

  • Block cytokines

  • Dual targeting of different disease mediators  

  • As delivery vehicles for payloads to targeted sites

Most bsAbs aim to treat cancer, but more are starting to target chronic inflammatory, autoimmune, and neurodegenerative diseases, as well as vascular, ocular, and hematologic disorders.

There are three main production methods:

  1. Quadroma technology: Relies on the somatic fusion of two different hybridoma cell lines. The random pairing of heavy and light chains creates bsAbs. BsAbs produced through quadroma technology are similar to conventional antibodies. Catumaxomab is the first approved bsAb and was produced via a rat/mouse quadroma cell line.

  2. Chemical conjugation: Site-specific chemical conjugation involves lysine or cysteine-based cross-linkers.Alternative methods include disulfide bridging, and incorporation of genetically encoded unnatural amino acids.

  3. Genetic approaches: Utilizes recombinant DNA technology. Genes encoding the variable domains of two different mAbs are cloned to produce a single construct. This results in the creation of bispecific formats such as scFv, diabody, or Fab.

There are currently 14 approved bsAb therapeutics worldwide, and >100 in clinical trials

Catumaxomab (Removab): Approved by EU in 2009; withdrawn in 2017. Anti-EpCAM and anti-CD3 trifunctional antibody. Treats malignant ascites.

Blinatumomab (Blincyto): Approved by US in 2014. BiTE bsAb targeting CD19 and CD3. Treats acute lymphoblastic leukemia.

Ozoralizumab (Nanozora): Approved in Japan in 2022. Trivalent, bispecific, humanized anti-TNFα antibody to treat rheumatoid arthritis. Binds to two subunits of TNFα to potently neutralize its action.

Elranatamab (Elrexfio): Accelerated approval by US in 2023. Humanized B-cell maturation antigen (BCMA)-CD3 bispecific antibody. Treats multiple myeloma.

Potential applications for bsAbs

Crossing the blood-brain barrier: Therapeutic antibodies may be re-engineered as a blood-brain barrier (BBB)-penetrating bispecific antibody. One arm would be the therapeutic antibody, and the other arm would be the transporting antibody. The transporting antibody would target an exofacial epitope on a BBB receptor, thus allowing for receptor-mediated transcytosis across the BBB.

Diagnostic assays: In an immunoassay, a monospecific capture antibody can be immobilized onto a solid surface to bind to a corresponding antigen in serum. Since bsAbs can function as a cross-linker, they can then be added to bind the captured antigen and reporter molecule.

Treatment of pathogens: BsAbs may be a solution to antimicrobial resistance (AMR). AMR occurs when pathogenic strains are no longer affected by multiple antibiotics or chemotherapeutic agents. DiGiandomenico et al. developed a bsAb, BiS2αPa, that targeted the bacterial pathogen Pseudomonas aeruginosa. The bsAbs conferred three mechanisms of action by binding with its two arms to Psl and PcrV. The study demonstrated the valuable potential of using multispecific antibody formats for the construction of therapeutic drugs.

Biointron focuses on antibody discovery, expression, and optimization. Contact us at +1 (732) 515-4766 or

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