What are bispecific antibodies?

A bispecific antibody drug (BsAb, BsMAb) is a synthetic protein capable of binding two distinct antigens, or two different epitopes on the same antigen, simultaneously. This dual specificity allows them to:

  • Induce protein complex interactions.
  • Interfere with receptor signalling.
  • Inactivate signalling ligands.
  • Activate immune cells.

BsAbs have been investigated for therapeutic applications in targeted drug delivery, Alzheimer’s disease, and cancer immunotherapy.

By recognising two epitopes, bispecific antibodies can:

  • Redirect T lymphocytes to tumour cells.
  • Inhibit two signalling pathways at once.
  • Target multiple disease agents simultaneously.
  • Deliver therapeutic payloads directly to specific sites.

Structure and Function

Monoclonal antibodies provide the foundation for BsAb design. Early strategies involved reducing and reoxidising hinge-region cysteines in monoclonal antibodies to form bispecific molecules. Today, BsAbs fall into two main structural classes:

  • IgG-based antibodies.
  • Variable fragment (Fv)-based antibodies.

Depending on therapeutic application, BsAbs are grouped into three categories:

  1. Antibodies targeting two tumour antigens.
  2. Antibodies targeting one tumour antigen and one phagocytic molecule.
  3. Antibodies targeting two immune-related molecules.

Learn more about Bispecific Antibody Drugs 

The BiTE Mechanism

Bi-specific T cell engagers (BiTEs) are a subclass of BsAbs developed as anti-cancer agents. They harness the host immune system by directing cytotoxic T cell activity against tumour cells. A typical BiTE binds both a tumour-associated antigen and the CD3 complex on T cells, and therefore falls under the second category of BsAbs.

Each BiTE molecule contains two single-chain variable fragments (scFvs):

  • Tumour-binding domain: targets antigens such as BCMA, CD19 or DLL3.
  • T cell-binding domain: targets CD3 on T cells.

The scFv is composed of fused variable regions of heavy (VH) and light (VL) antibody chains, linked with a flexible peptide. Two scFvs joined by a linker create the dual-binding domains of a BiTE. Importantly, the tumour-targeting scFv can be adapted to different antigens, enabling off-the-shelf therapeutic flexibility.

Unlike natural antibodies, BiTEs directly connect T cells to tumour cells. This leads to T cell activation, proliferation, and enhanced tumour lysis, all without co-stimulation or MHC presentation²³.

Activated T cells then release cytotoxic proteins, including perforin and granzymes, which form pores in tumour cell membranes and trigger apoptosis.

BiTe-Targets1

Clinical Significance of BiTEs

Natural antibodies cannot directly recruit T cells, as T cells lack Fc receptors. By binding CD3, BiTEs bypass this limitation, enabling T cell activation and proliferation via antigen-specific signalling³⁴.

Key features of BiTE-driven T cell activation include:

  • Requirement of tumour cell presence for T cell activation.
  • Independence from co-stimulatory signals such as CD28 or IL-2.
  • Reactivation of exhausted T cells exposed to tumour antigens.

The first BiTE therapy approved for clinical use was blinatumomab, which targets CD19 on both healthy and malignant B cells. It is effective at very low concentrations (10–100 pg/ml), inducing serial target-cell lysis in patients with acute lymphoblastic leukaemia (ALL) and non-Hodgkin lymphoma⁵⁶.

Compared to monoclonal antibody (mAb) drugs or combinations, BsAbs offer:

  • Higher binding avidity.
  • Greater cytotoxicity.
  • Reduced drug resistance.
  • Novel functionalities arising from their unique structure.

Clinical acceptance of BiTE antibody drugs

To date, nine bispecific antibodies have received global approval, including five FDA-approved agents:

  • Blinatumomab
  • Emicizumab
  • Amivantamab
  • Tebentafusp-tebn
  • Faricimab-svoa
BiTE-antibodies-approved-list

Bispecific antibody drug research

Developing and optimising BiTEs requires careful bioanalytical assessment. Accurate pharmacological quantification in vitro helps:

  • Improve the therapeutic index of candidate drugs.
  • Identify the most promising compounds early in discovery.
  • Predict safe, pharmacologically active starting doses for clinical studies.

This is a key challenge in developing CD3-bispecific therapies.

Krishgen is the only commercial manufacturer currently offering ELISA kits specifically for bispecific antibody pharmacokinetic studies. With 40+ drug targets available in highly sensitive sandwich ELISA format, Krishgen supports BsAb research with robust analytical tools.

Example assays:

  • Teclistamab: microwells pre-coated with recombinant BCMA protein; HRP-conjugated CD3 as detector.
  • Blinatumomab: microwells pre-coated with CD19 protein; HRP-conjugated CD3 as detector.

Find an ELISA

References

  • Einsele, H. et al. The BiTE (bispecific T-cell engager) platform: development and future potential of a targeted immuno-oncology therapy across tumour types. Cancer126, 3192–3201 (2020). doi:10.1002/cncr.32909

  • Amgen. The shape of drugs to come: bispecific antibodies. Available at: https://www.amgen.com/stories/2018/08/the-shape-of-drugs-to-come/bispecific-antibody (accessed 2024).

  • Tian, Z., Liu, M., Zhang, Y. et al. Bispecific T cell engagers: an emerging therapy for management of haematologic malignancies. J. Hematol. Oncol.14, 75 (2021). doi:10.1186/s13045-021-01084-4

  • Biochempeg. Approved bispecific antibody drugs. Available at: www.biochempeg.com/article/71.html (accessed 2024).

  • Brennan, M., Davison, P. F. & Paulus, H. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science229, 81–83 (1985).

  • Engelberts, P. J. et al. DuoBody-CD3xCD20 induces potent T-cell-mediated killing of malignant B cells in preclinical models and provides opportunities for subcutaneous dosing. EBioMedicine52, 102625 (2020).

  • Nagorsen, D. & Baeuerle, P. A. Immunomodulatory therapy of cancer with T cell-engaging BiTE antibody blinatumomab. Exp. Cell Res.317, 1255–1260 (2011).

  • Brischwein, K. et al. Strictly target cell-dependent activation of T cells by bispecific single-chain antibody constructs of the BiTE class. J. Immunother.30, 798–807 (2007).

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Krishgen Biosystems is an original manufacturer of therapeutic antibody ELISA kits, HCP detection assays, immunotherapy assay kits, antibodies, proteins, assays for drug development.

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