Bispecific antibodies (bsAbs) are engineered immunoglobulins that incorporate two antigen-binding sites specific for distinct epitopes, typically located on two separate antigens. One binding specificity is generally designed to target a particular cell surface antigen on the target cell, while the other binds to a "triggering" molecule on the surface of an effector cell, such as Fcγ receptors or the CD3/T-cell receptor complex.

This dual specificity allows bispecific antibodies to redirect the activity of effector cells toward target cells that would otherwise evade immune detection. Different effector cells express various triggering molecules (receptors), enabling diverse cytotoxic responses by altering the binding domains. Additionally, by directing one binding domain to serum immunoglobulin, bispecific antibodies can leverage a range of effector functions, including antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, complement activation, and extended serum half-life.

Currently, two bispecific antibodies are approved for therapeutic use, with a growing number undergoing clinical trials for indications such as cancer and other diseases, reflecting their unique mechanisms of action and expanding interest in their therapeutic potential.

Advantages and Limitations of Bispecific Antibodies

Advantages:

• Bispecific antibodies enhance cytotoxicity by directing effector cells to tumor cells.

• They can simultaneously recognize two molecular targets, improving selectivity, functional affinity, safety, and therapeutic efficacy.

• Development and clinical trial costs are reduced compared to combination therapy with two monoclonal antibodies.

Limitations:

• Challenges in manufacturing due to potential mismatching between heavy and light chains.

• Non-natural structures may induce immunogenicity.

Structure and Formats of Bispecific Antibodies

The modular architecture of antibodies has facilitated the development of over 100 bispecific antibody formats, differing in attributes such as molecular weight, number and spatial arrangement of antigen-binding sites, valency, immune function support, and pharmacokinetics.

Classification:

1. Fc-Containing Bispecific Antibodies:

   • These retain Fc-mediated effector functions such as complement-dependent cytotoxicity (CDC) and ADCC.

   • Examples include "knob-into-hole" IgG, crossMAb, ortho-Fab IgG, DVD-Ig, two-in-one IgG, IgG-scFv, and scFv2-Fc formats.

2. Non-Fc Bispecific Antibodies:

   • These lack Fc regions, offering improved tumor penetration due to smaller size.

   • Formats include tandem scFvs, diabodies, single-chain diabodies, tandem diabodies (TandAbs), dual-affinity retargeting molecules (DARTs), dock-and-lock (DNL) structures, and nanobodies.

Production of Bispecific Antibodies

Numerous methods have been developed to generate bispecific antibodies:

1. Hybrid Hybridoma (Quadroma):

   • An early approach relying on the somatic fusion of two hybridoma cell lines producing murine IgGs with desired specificities. However, the yield of functional bispecific antibodies is unpredictable, requiring extensive purification.

2. Recombinant Techniques:

   • Bispecific IgG antibodies are assembled from distinct heavy and light chains expressed in the same production system. Typically, at least two plasmids are required for heterodimerized heavy chains, alongside one plasmid for a common light chain or two plasmids for separate light chains.

   • Expressing heavy chains (HC) and light chains (LC) on separate plasmids allows for plasmid ratio manipulation, optimizing assembly and production efficiency. Selection of stable clonal cell lines is labor-intensive and time-consuming but necessary for large-scale manufacturing.

3. Transient Transfection:

   • Offers rapid results without genomic integration, enabling efficient early-stage development. Human embryonic kidney (HEK293) and HEK-based Expi293 cells are commonly employed for transient expression in bispecific antibody development.

By leveraging expertise in antibody engineering and biomanufacturing, significant advancements have been achieved in optimizing bispecific antibody production processes, ensuring high yields and functionality for therapeutic applications.

CliniSciences Production Scientists have the experience in producing a variety of IgG formats including Bispecific IgG.

Please contact us at tech@clinisciences.com for more information or for a quote request.

References

1. Subramanian, G. (Ed.). (2013). Antibodies: Volume 1: Production and Purification. Springer Science & Business Media.
2. Marrocco, I., Romaniello, D., & Yarden, Y. (2019). Cancer immunotherapy: the dawn of antibody cocktails. In Human Monoclonal Antibodies (pp. 11-51). Humana Press, New York, NY.
3. Sedykh, S. E., Prinz, V. V., Buneva, V. N., & Nevinsky, G. A. (2018). Bispecific antibodies: design, therapy, perspectives. Drug design, development and therapy, 12, 195.
4. Kontermann, R. (2012, March). Dual targeting strategies with bispecific antibodies. In MAbs (Vol. 4, No. 2, pp. 182-197). Taylor & Francis.
5. Brinkmann, U., & Kontermann, R. E. (2017, February). The making of bispecific antibodies. In MAbs (Vol. 9, No. 2, pp. 182-212). Taylor & Francis.
6. Spiess, C., Zhai, Q., & Carter, P. J. (2015). Alternative molecular formats and therapeutic applications for bispecific antibodies. Molecular immunology, 67(2), 95-106.
7. Zhang, X., Yang, Y., Fan, D., & Xiong, D. (2017). The development of bispecific antibodies and their applications in tumor immune escape. Experimental hematology & oncology, 6(1), 12.