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Bispecific antibodies: a mechanistic review of the pipeline


The term bispecific antibody (bsAb) is used to describe a large family of molecules designed to recognize two different epitopes or antigens. BsAbs come in many formats, ranging from relatively small proteins, merely consisting of two linked antigen-binding fragments, to large immunoglobulin G (IgG)-like molecules with additional domains attached. An attractive bsAb feature is their potential for novel functionalities — that is, activities that do not exist in mixtures of the parental or reference antibodies. In these so-called obligate bsAbs, the physical linkage of the two binding specificities creates a dependency that can be temporal, with binding events occurring sequentially, or spatial, with binding events occurring simultaneously, such as in linking an effector to a target cell. To date, more than 20 different commercialized technology platforms are available for bsAb creation and development, 2 bsAbs are marketed and over 85 are in clinical development. Here, we review the current bsAb landscape from a mechanistic perspective, including a comprehensive overview of the pipeline.

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The authors thank R. Roovers and H. van der Vliet for helpful comments on the manuscript and A. Cook and V. P. Rath for access to the Beacon Targeted Therapies database.

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All authors contributed substantially to all aspects of the article.

Correspondence to Paul W. H. I. Parren.

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Competing interests

A.F.L. and M.L.J. are employees of Genmab, a biotechnology company that develops therapeutic antibodies including bispecific antibodies and bispecific antibody technology. They own warrants and/or stock. P.W.H.I.P. is an employee of Lava Therapeutics, a start-up biotechnology company that develops therapeutic antibodies including bispecific antibodies and bispecific antibody technology. He obtains stock options as part of his employment.

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Combinatorial bsAbs

Bispecific antibodies (bsAbs) that display an activity or functionality that can also be obtained by combining separate antibodies with the same specificities (for example, a parental or reference antibody mixture).

Obligate bsAbs

Bispecific antibodies (bsAbs) that display an activity or functionality that is dependent on the physical linkage of the two specificities (and cannot be obtained by combining separate antibodies with the same specificities). The dual-targeting concepts mediated by these bsAbs are considered obligate concepts.

Chain-association issue

The co-expression of two different heavy (H) and two different light (L) chains results in a complex mixture of sixteen possible H2L2 recombinations, representing ten different antibodies. Only one of these antibodies (represented by two possible H2L2 recombinations) corresponds with the desired bispecific antibody (maximal yield 12.5% in the mixture). This issue is addressed by strategies forcing cognate HL-pairing and/or promoting heterodimerization of the two different H chains.


The number of antigen-binding sites in an antibody molecule. The design of a bispecific antibody (bsAb) format influences the number of binding sites per target. A bivalent bsAb with one binding site for each target is denoted as 1 + 1. Incorporating additional binding sites can lead to trivalent (2 + 1) and tetravalent (2 + 2 or 1 + 3) designs.

Antibody fragments

The antibody molecule consists of different domains that can be expressed separately and used as modular building blocks. The domains involved in antigen recombination are often used as binding moieties in the design of antibody-based therapeutics. Examples include domain antibodies (heavy chain-only variable domain (VHH)) and single-chain Fv fragments (scFvs), antigen-binding fragments (Fabs), single-chain Fab fragments (scFabs) and, more recently, single-chain Fc fragments (scFcs).

Cross-arm binding efficiency

An increase in apparent affinity when a bispecific antibody binds to the second target or receptor following its binding to the first target or receptor on the same cell.

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Fig. 1: Timeline of conceptual and technical innovations contributing to the development of the therapeutic bsAb landscape.
Fig. 2: A selection of bsAb formats.
Fig. 3: Overview of the clinical development pipeline for bsAbs.
Fig. 4: Year of clinical study initiation for bsAbs.
Fig. 5: Examples of obligate mechanisms of action of bsAbs.