Antibody-based drugs have proven effective against infectious and chronic inflammatory diseases, cancer, and more. Advances in recombinant engineering strategies to impart multi-specificity to these already powerful molecules continue to expand the therapeutic potential of antibodies. As such, antibodies with dual specificity or bispecific antibodies have emerged as a significant modality in the evolution of antibody drugs. Bispecific antibodies are engineered molecules designed to bind two distinct epitopes within an antigen or two completely different antigens.
Bispecific antibodies enable novel mechanisms of action and improved potency, unachievable by combined treatment with monoclonal antibodies. Several mechanisms of action, including cross-linking of mitogen receptors, localized recruitment of immune cells, and capacity to cross the blood-brain barrier, make bispecific antibodies valuable drugs to reduce tumor growth, improve tumor cell killing, and target traditionally inaccessible tumors.1,2 Additionally, their dual specificity makes it possible to target redundant signaling pathways, reducing the chance of treatment resistance. Lastly, careful selection of antigen specificities enables controlled targeting of diseased cells, safeguarding healthy cells and tissues and thus reducing the potential of adverse effects.2
The first clinically used bispecific antibody, Catumaxomab targeting EpCAM and CD3ε, was approved in 2009 to treat a cancer-related condition. Despite their therapeutic potential, the progression of these molecules to the clinic was first limited by existing technological and production capabilities, with only three bispecific antibody drugs approved by 2020.3 Nevertheless, huge leaps in technologies and platforms for the development and production of these molecules, as well as a greater understanding of their therapeutic mechanisms and disease-related targets of significance, have boosted their therapeutic availability. About eleven bispecific antibody drugs have been approved globally since 2020 and several are pending or undergoing review.4 The majority of approved bispecific antibodies to date target cancer-relevant molecules, with T-cell engagers having dual specificity for CD3 and tumor antigens (e.g., BCMA, gp100, and CD20) representing a frequent modality. Epcoritamab (CD20/CD3), Glofitamab (CD20, CD3ε), and Talquetamab (GPCR5D/CD3) are all the T-cell engagers approved for clinical use in 2023.4
Bispecific antibodies may be engineered as IgG-like or non-IgG-like molecules to bind specific antigens of therapeutic significance. A critical distinction between these two formats is the constant Fc fragment in IgG-like bispecific antibodies, which confers various effector functions such as complement activation (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC).5 Therefore, this format offers expanded therapeutic potential beyond antigen-neutralization. Additionally, the larger molecular size of IgG-like bispecific antibodies helps their stability and extends plasma half-life, promoting therapeutic efficacy.
Bispecific antibody mechanisms of action. A. In-trans cell binding establishes a connection between cells, facilitating cytotoxicity. B. In-cis receptor agonism brings receptors together, activating signaling. C. In-cis receptor antagonism blocks receptor interaction, inhibiting signaling. D. Piggybacking enables receptor-mediated transcytosis and reaching specific intracellular targets. E. Localization effects help direct therapeutic function to the right cells and tissues, reducing adverse effects. F. Co-factor mimetics bring together substrates and enzymes to promote specific enzymatic reactions.6 Figure 1, retrieved without modification from Madsen, et al. 2024.6 https://creativecommons.org/licenses/by/4.0/
In contrast, the therapeutic activity of non-IgG-like bispecific antibodies is limited to their unique antigen-binding properties, as they lack effector functions. Overall, their smaller size has both negative and positive implications for their effectiveness as therapeutics. For instance, these smaller molecules tend to have faster serum clearance, which limits efficacy. Yet, their small size facilitates their production, favors tissue penetration, and reduces immunogenicity risks.5
A critical challenge in efficiently producing IgG-like bispecific antibodies is achieving the desired combination of heavy and light chains, resulting in a functional heterodimeric antibody. To date, several platforms leveraging novel protein engineering strategies have been developed to ensure the desired chain pairing and reduce the generation of spurious antibodies when developing IgG-like asymmetric and symmetric or non-IgG-like bispecific antibodies. 7,8
To develop IgG-like asymmetric bispecific antibodies, the Fc domain may be modified to induce specific structural rearrangements (Knobs-Into-Holes), introduce complementary domains (SEEDbody), or add charged residues to help promote heavy chain heterodimerization (ART-Ig).8 Although technologies such as Knobs-Into-Holes can efficiently reduce heavy chain mispairing, “Knob” and “Hole” heavy chains must be similarly expressed to ensure reduced homodimerization.6
Diagrams of bispecific antibody formats were retrieved from Figure 1 of Ma et al. 2021. https://creativecommons.org/licenses/by/4.0/
CrossMab and DuoBody platforms for asymmetric bispecific antibody development help tackle light chain mispairing. In CrossMab, the constant (CH1-CL) or variable (VH-VL) domains are flipped in one Fab to ensure correct heavy/light chain pairing.11 The DuoBody platform takes advantage of the IgG4 naturally occurring process known as Fab arm exchange. In this process, the reduction of hinge region disulfide bonds prompts the dissociation of the IgG4 molecule into two halves for random pairing upon disulfide bond oxidation. The DuoBody approach first introduces different single Fc mutations on each monoclonal antibody intended for heterodimerization. Following mixing and the dissociation of modified monoclonal antibody pairs under reducing conditions, the recombination of Fc arms carrying different mutations and specificities or heterodimerization is favored, a process referred to as controlled Fab arm exchange.5
Other platforms support generating symmetric IgG-like bispecific antibodies by adding variable domains (VL/VH) or Fab fragments that impart additional specificity and valency to a scaffold IgG monoclonal antibody (i.e., DVD-Ig and FIT-Ig).8 The DVD-Ig platform simplifies manufacturing and purification processes while ensuring correct chain pairing and bifunctionality.11
Non-IgG-like bispecific antibodies are generally simpler structures consisting of variable domains or single-chain variable fragments (scFvs).2 Because they lack the Fc antibody region, non-IgG-like bispecific antibodies are small molecules with short serum half-life. Therefore, engineering strategies aim to develop larger structures by either multimerizing various antibody fragments with peptide linkers or conjugation to larger molecules, such as human serum albumin and polyethylene glycol (PEG).11
Diagrams of bispecific antibody formats were retrieved from Figure 1 of Runcie et al. 2018. https://creativecommons.org/licenses/by/4.0/
1. Dietrich, S., Gross, A. W., Becker, S., Hock, B., Stadlmayr, G., Rüker, F., & Wozniak-Knopp, G. (2020). Constant domain-exchanged Fab enables specific light chain pairing in heterodimeric bispecific SEED-antibodies. Biochimica et Biophysica Acta - Proteins and Proteomics, 1868(1). https://doi.org/10.1016/j.bbapap.2019.07.003
2. Xu, Y., Lee, J., Tran, C., Heibeck, T. H., Wang, W. D., Yang, J., Stafford, R. L., Steiner, A. R., Sato, A. K., Hallam, T. J., & Yin, G. (2015). Production of bispecific antibodies in “knobs-into-holes” using a cell-free expression system. MAbs, 7(1). https://doi.org/10.4161/19420862.2015.989013
3. Surowka, M., & Klein, C. (2024). A pivotal decade for bispecific antibodies? MAbs, 16(1). https://doi.org/10.1080/19420862.2024.2321635
4. Antibody therapeutics product data. The Antibody Society. (2023, November 1). https://www.antibodysociety.org/antibody-therapeutics-product-data/
5. Abdeldaim, D. T., & Schindowski, K. (2023). Fc-Engineered Therapeutic Antibodies: Recent Advances and Future Directions. In Pharmaceutics (Vol. 15, Issue 10). https://doi.org/10.3390/pharmaceutics15102402
6. Madsen, A. v., Pedersen, L. E., Kristensen, P., & Goletz, S. (2024). Design and engineering of bispecific antibodies: insights and practical considerations. In Frontiers in Bioengineering and Biotechnology (Vol. 12). https://doi.org/10.3389/fbioe.2024.1352014y/articles/10.3389/fimmu.2021.626616/full
7. Gong, S., & Wu, C. (2019). Generation of Fabs-in-tandem immunoglobulin molecules for dual-specific targeting. Methods, 154. https://doi.org/10.1016/j.ymeth.2018.07.014
8. Ma, J., Mo, Y., Tang, M., Shen, J., Qi, Y., Zhao, W., Huang, Y., Xu, Y., & Qian, C. (2021). Bispecific Antibodies: From Research to Clinical Application. In Frontiers in Immunology (Vol. 12). https://doi.org/10.3389/fimmu.2021.626616
9. Carter, P., Ridgway, J. B. B., & Presta, L. G. (1996). ‘Knobs-into-holes’ provides a rational design strategy for engineering antibody CH3 domains for heavy chain heterodimerization. Immunotechnology, 2(1). https://doi.org/10.1016/1380-2933(96)80685-3
10. Yanakieva, D., Pekar, L., Evers, A., Fleischer, M., Keller, S., Mueller-Pompalla, D., Toleikis, L., Kolmar, H., Zielonka, S., & Krah, S. (2022). Beyond bispecificity: Controlled Fab arm exchange for the generation of antibodies with multiple specificities. MAbs, 14(1). https://doi.org/10.1080/19420862.2021.2018960
11. Li, H., Er Saw, P., & Song, E. (2020). Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics. In Cellular and Molecular Immunology (Vol. 17, Issue 5). https://doi.org/10.1038/s41423-020-0417-8
12. Jakob, C. G., Edalji, R., Judge, R. A., DiGiammarino, E., Li, Y., Gu, J., & Ghayur, T. (2013). Structure reveals function of the dual variable domain immunoglobulin (DVD-IgTM) molecule. MAbs, 5(3). https://doi.org/10.4161/mabs.23977
13. Runcie, K., Budman, D. R., John, V., & Seetharamu, N. (2018). Bi-specific and tri-specific antibodies- the next big thing in solid tumor therapeutics. In Molecular Medicine (Vol. 24, Issue 1). https://doi.org/10.1186/s10020-018-0051-4
