Introduction

FDA-Approved Bispecific Antibodies

As of December 2023, the U.S. Food and Drug Administration (FDA) has approved 11 bispecific antibody (bsAb) products, primarily targeting cancer, hematologic conditions, and ocular diseases^[1]. The development of bsAbs is rapidly growing, with over 100 bsAbs currently in clinical development^[2].

Key milestones in bsAb development include the approval of Blinatumomab, a CD19/CD3 T-cell engaging bsAb for B-cell precursor acute lymphoblastic leukemia (ALL), which has shown significant efficacy in clinical trials compared to traditional chemotherapy^[3]. Another notable approval is Amivantamab, targeting EGFR and cMET for non-small cell lung cancer (NSCLC), demonstrating the potential of bsAbs to enhance therapeutic efficacy by targeting multiple pathways simultaneously^[4].

Below is a detailed list of the FDA-approved bispecific antibodies^{[2] [5] [6]}:

Table 1. FDA-Approved Bispecific Antibodies

Despite these successes, the development and approval process for bsAbs remains complex, requiring rigorous preclinical and clinical evaluations to ensure safety and efficacy^[7].

Bispecific Antibody Formats

With over 100 different formats available, bsAbs provide a wide range of therapeutic possibilities. Each format has unique advantages and challenges, making the selection of the appropriate format crucial for specific therapeutic goals. Bispecific antibodies can be broadly categorized into two main types based on the presence or absence of the Fc region: IgG-like bsAbs and non-IgG-like bsAbs.

IgG-like Bispecific Antibodies

IgG-like bsAbs contain an Fc region, which provides several advantages for manufacturing and clinical applications. The Fc region stabilizes antibodies in the bloodstream, prolonging their half-life and facilitating purification^[8]. However, the inclusion of the Fc region can also affect the biological activity and affinity of the bsAbs, complicating their analysis and detection^[8]. These bsAbs can be further categorized into asymmetry and symmetry formats:

• Asymmetry Formats: These bsAbs feature two distinct antigen-binding domains and can be engineered to have different specificities, affinities, or functions for each antigen. This flexibility allows them to target diverse antigens and modulate specific biological pathways or signaling mechanisms^[9].
• Symmetry Formats: These bsAbs are designed with two identical antigen-binding domains and a single Fc region. This format enhances manufacturability and stability, and due to the presence of the Fc region, it can mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC)^[9].

Non-IgG-like Bispecific Antibodies

Non-IgG-like bsAbs, also known as fragment formats, lack the Fc region and are constructed by combining two different monoclonal antibody fragments, such as single-chain variable fragments (scFvs) or antigen-binding fragments (Fab). The absence of an Fc fragment provides non-IgG-like bsAbs with unique advantages, including easier production in both eukaryotic and prokaryotic expression systems and smaller molecular masses that facilitate tissue penetration and rapid localization to specific antigen-binding sites[8]. However, the lack of an Fc region also presents certain drawbacks, such as rapid drug clearance from the body and a shorter blood half-life^[7].

Expression Systems for Bispecific Antibody Production

Producing bispecific antibodies (bsAbs) needs advanced expression systems to accurately fold and modify these complex molecules. The choice of expression system affects the yield, stability, and functionality of bsAbs. Common expression systems include mammalian cells, bacteria, yeast, etc.

Mammalian Expression Systems

Mammalian cells are widely used for producing therapeutic antibodies, including bsAbs, due to their complex post-translational modifications that resemble human proteins. This reduces immunogenicity and ensures proper folding and functionality^[10].

Chinese Hamster Ovary (CHO) Cells

• Characteristics: CHO cells are well-characterized and yield high protein quantities. They are ideal for producing complex recombinant proteins^[11].
• Advantages: High yield, stable transfection, and excellent biosafety^[11].
• Challenges: Slower growth, impacting production timelines^[11].

Human Embryonic Kidney 293 (HEK293) Cells

• Characteristics: HEK293 cells have high transfection efficiency and scalable production capabilities. They provide a human-like environment for protein folding^[12].
• Advantages: High transfection efficiency, scalable production, suitable for early-stage research^[12].
• Challenges: Requires optimization for large-scale production^[13].

Bacterial Expression Systems

Bacterial systems, especially E. coli, are used for producing smaller antibody fragments like scFvs.

Escherichia coli (E. coli)

• Characteristics: E. coli has fast growth, low cost, and high protein yield, making it suitable for early-stage research^[14].
• Advantages: Rapid growth, low cost, high yield^[14].
• Challenges: Lack of post-translational modification machinery, limiting its use for full-length antibodies^[14].

Yeast Expression Systems

Yeast systems balance rapid growth with some post-translational modifications^[15].

accharomyces cerevisiae

• Characteristics: Yeast cells grow rapidly and can produce large quantities of antibodies^[16]. They combine prokaryotic advantages with some post-translational modifications^[17].
• Advantages: Rapid growth, high yield, ability to perform some post-translational modifications^[15].
• Challenges: Differences in intracellular environments can pose challenges for complex antibody folding^[17].

Trends and challenges of Bispecific Antibodies

Despite their significant therapeutic potential, bispecific antibodies (bsAbs) face several challenges that need to be addressed to fully realize their clinical benefits.

New Target Exploration

Researchers are actively exploring new targets to expand the therapeutic applications of bsAbs. Targets such as B7-H4, ENPP3, and CLDN6 are being investigated to enhance the efficacy and broaden the range of diseases that can be treated with bsAbs^[18]. Validating these new targets through rigorous in vitro and in vivo experiments is crucial for their clinical success. For instance, JNJ-78306358, a first-in-class bispecific T cell engaging antibody targeting CD3 and HLA-G, has shown promising results in preclinical studies, demonstrating the potential of targeting novel antigens^[18].

Technological Breakthroughs

Advancements in antibody engineering are continuously improving the stability and production efficiency of bsAbs. Techniques such as CrossMab and knobs-into-holes are enhancing the manufacturability and functionality of these complex molecules^[19]. Novel formats like Nanobodies and TandAbs are also being developed to improve tissue penetration and specificity^[19]. These technological breakthroughs are essential for overcoming the inherent challenges associated with the production and application of bsAbs.

Production Challenges

Producing bsAbs involves intricate recombinant DNA techniques and cell culture processes. Optimizing these processes to improve yield and reduce costs is a key area of research. Ensuring the stability and consistency of bsAbs during production is another critical challenge^[19]. Recent advancements in expression vector design and cell-line development have shown promise in addressing these issues, paving the way for industrial-scale production^[19]. For example, KBI Biopharma's innovative approach has achieved up to 99% heterodimers, significantly enhancing the efficiency of bsAb production.

Safety and Side Effects

BsAbs may induce severe side effects, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS)^[19]. Researchers are working on engineering strategies and clinical trials to mitigate these adverse effects and improve the safety profile of bsAbs^[19]. For example, pre-existing reactivity screening and characterization during the preclinical phase can help mitigate immunogenicity risks^[19].

Conclusion

The development of bispecific antibodies (bsAbs) has ushered in a new era of precision medicine, with the potential to revolutionize the treatment of various diseases through their unique ability to engage multiple targets simultaneously. While significant progress has been made in the design, production, and clinical application of bsAbs, several challenges remain. Addressing these challenges will require continued innovation in target identification, antibody engineering, and manufacturing processes.As research advances and new technologies emerge, the field is poised to overcome current limitations and expand the therapeutic potential of bsAbs.

References

[1] Lim, K., Zhu, X. S., Zhou, D., Ren, S., & Phipps, A. (2024). Clinical Pharmacology Strategies for Bispecific Antibody Development: Learnings from FDA-Approved Bispecific Antibodies in Oncology. Clinical Pharmacology and Therapeutics , 116 (2), 315–327. https://doi.org/10.1002/cpt.3308

[2] U.S. FDA. Bispecific antibodies: area of research and clinical applications. Retrieved from https://www.fda.gov/drugs/spotlight-cder-science/bispecific-antibodies-area-research-and-clinical-applications

[3] Przepiorka, D., Ko, C. W., Deisseroth, A., Yancey, C. L., Candau-Chacon, R., Chiu, H. J., Gehrke, B. J., Gomez-Broughton, C., Kane, R. C., Kirshner, S., Mehrotra, N., Ricks, T. K., Schmiel, D., Song, P., Zhao, P., Zhou, Q., Farrell, A. T., & Pazdur, R. (2015). FDA Approval: Blinatumomab. Clinical Cancer Research , 21 (18), 4035–4039. https://doi.org/10.1158/1078-0432.CCR-15-0612

[4] Chon, K., Larkins, E., Chatterjee, S., Mishra-Kalyani, P. S., Aungst, S., Wearne, E., Subramaniam, S., Li, Y., Liu, J., Sun, J., Charlab, R., Zhao, H., Saritas-Yildirim, B., Bikkavilli, R. K., Ghosh, S., Philip, R., Beaver, J. A., & Singh, H. (2023). FDA Approval Summary: Amivantamab for the Treatment of Patients with Non-Small Cell Lung Cancer with EGFR Exon 20 Insertion Mutations. Clinical Cancer Research , 29 (17), 3262–3266. https://doi.org/10.1158/1078-0432.CCR-22-3713

[5] Elrexfio. Retrieved from https://www.elrexfio.com/

[6] Talvey. Retrieved from https://www.talvey.com/

[7] Goebeler, M. E., Stuhler, G., & Bargou, R. (2024). Bispecific and multispecific antibodies in oncology: opportunities and challenges. Nat Rev Clin Oncol , 21 , 539–560. https://doi.org/10.1038/s41571-024-00905-y

[8] Labrijn, A. F., Janmaat, M. L., Reichert, J. M., et al. (2019). Bispecific antibodies: a mechanistic review of the pipeline. Nat Rev Drug Discov , 18 , 585–608. https://doi.org/10.1038/s41573-019-0028-1

[9] Brandão, R. O., Jiang, X., Selvaraju, S., & Mohapatra, P. (2024). Bispecific Antibodies: An Emerging Concept in Antibody-Based Cancer Therapies. In: Das Mohapatra, A., Sahu, P. S. (eds) Advances in Immunology and Immuno-techniques. Interdisciplinary Biotechnological Advances . Springer, Singapore. https://doi.org/10.1007/978-981-97-5508-0_4

[10] Frenzel, A., Hust, M., & Schirrmann, T. (2013). Expression of recombinant antibodies. Front Immunol , 4 , 217. https://doi.org/10.3389/fimmu.2013.00217

[11] Wang, C., Guo, X., Wang, W., Li, J.-X., & Wang, T.-Y. (2025). From Cell Clones to Recombinant Protein Product Heterogeneity in Chinese Hamster Ovary Cell Systems. International Journal of Molecular Sciences , 26 (3), 1324. https://doi.org/10.3390/ijms26031324

[12] Pulix, M., Lukashchuk, V., Smith, D. C., & Dickson, A. J. (2021). Molecular characterization of HEK293 cells as emerging versatile cell factories. Current Opinion in Biotechnology , 71 , 18–24. https://doi.org/10.1016/J.COPBIO.2021.05.001

[13] Zhang, L., Gao, J., Zhang, X., Wang, X., Wang, T., & Zhang, J. (2024). Current strategies for the development of high-yield HEK293 cell lines. Biochemical Engineering Journal , 205 , 109279. https://doi.org/10.1016/J.BEJ.2024.109279

[14] İncir, İ., & Kaplan, Ö. (2024). Escherichia coli as a versatile cell factory: Advances and challenges in recombinant protein production. Protein Expression and Purification , 219 , 106463. https://doi.org/10.1016/J.PEP.2024.106463

[15] Mustafa, M. I., Alzebair, A. A., & Mohammed, A. (2024). Development of Recombinant Antibody by Yeast Surface Display Technology. Current Research in Pharmacology and Drug Discovery , 6 , 100174. https://doi.org/10.1016/J.CRPHAR.2024.100174

[16] Maneira, C., Chamas, A., & Lackner, G. (2025). Engineering Saccharomyces cerevisiae for medical applications. Microb Cell Fact , 24 , 12. https://doi.org/10.1186/s12934-024-02625-5

[17] Li, Y., Wang, X., Zhou, N. Y., & Ding, J. (2024). Yeast surface display technology: Mechanisms, applications, and perspectives. Biotechnology Advances , 76 , 108422. https://doi.org/10.1016/J.BIOTECHADV.2024.108422

[18] Obermajer, N., Zwolak, A., van de Ven, K., Versmissen, S., Menard, K., Rogers, K., Petley, T., Weinstock, D., Aligo, J., Patel, J., Tian, K., Angelillo, L., Yi, F., Jarantow, S., Schutsky, K., Hamuro, Y., Arias, D. A., Buyens, K., Sheena Yao, T.-W., ... Laquerre, S. (2025). JNJ-78306358, a First-in-Class Bispecific T Cell Engaging Antibody targeting CD3 and HLA-G. iScience , 111876. https://doi.org/10.1016/J.ISCI.2025.111876

[19] Karbyshev, M. S., Kalashnikova, I. V., Dubrovskaya, V. V., Baskakova, K. O., Kuzmichev, P. K., & Sandig, V. (2025). Trends and challenges in bispecific antibody production. Journal of Chromatography A , 1744 , 465722. https://doi.org/10.1016/J.CHROMA.2025.465722