Bispecific Antibody Overview

What are Bispecific Antibodies

Bispecific antibodies (bsAbs) are antibodies containing two antigen-binding sites for different epitopes (usually on two separate antigens). Typically one binding specificity will be directed against a specific cell surface antigen of the target cell and the other against a 'triggering' molecule on the surface of the effector cell e.g. one of the FcyR or the CD3 / T cell receptor complex.

The bispecific antibody can override the specificity of an effector cell for its natural target and redirect it to kill a target that it would otherwise ignore. Different cytotoxic cells express different triggering molecules (receptors). Thus, by varying the specificities of target and effector binding domains a variety of effector responses can be directed against most types of target cells. Alternatively, the full range of effector functions (i.e. ADCC, phagocytosis, complement activation and extended serum half-life) can be conferred by targeting one binding specificity to serum immunoglobulin.

Currently, there are two bispecific antibodies for therapeutic use in the market. Due to their unique mechanism of action, bispecific antibodies are gaining a lot of interest and more bispecific antibodies are under clinical trials not only for cancers, but also for other diseases.

How do bispecific monoclonal antibodies work
Fig 1. How do bispecific monoclonal antibodies work

Bispecific Antibody Structure and Formats

There are more than 100 different bispecific antibody formats have been produced due to the modular architecture of antibodies. These formats vary in many ways including their molecular weight, number of antigen-binding sites, spatial relationship between different binding sites, valency for each antigen, ability to support secondary immune functions and pharmacokinetic half-life.

Recombinant bispecific antibodies can be divided into two classes: bispecific formats with Fc regions, and bispecific formats without Fc regions. Bispecific antibodies with an Fc region retain Fc-mediated effector functions, such as CDC and ADCC. These formats roughly include "knob into hole" IgG, crossMab, ortho-Fab IgG, DVD-Ig, two in one IgG, IgG-scFv and scFv2-Fc.

Bispecific antibodies with no Fc lack Fc-mediated effector functions. However, the smaller size of such antibodies offers a better tumor tissue penetration over IgG-like formats. In this format, the variable domains of each parental monoclonal antibody and the linkers are cloned and linked to form a single-chain bispecific antibody. These bispecific antibodies represent many formats, including tandem scFvs, diabody format, single-chain diabodies, tandem diabodies (TandAbs), dual-affinity retargeting molecules (DARTs), dock-and-lock (DNL), and nanobodies.

Combining our rich experience in both antibody engineering and manufacturing, Sino Biological has great capability in bispecific antibody production with sufficiently high yields.

Common bispecific antibodies formats
Fig 2. Common bispecific antibodies formats

How to Make Bispecific Antibodies

A number of strategies have been developed to generate bispecific antibodies. Hybrid hybridoma (also referred to as quadroma) was the earliest technology used to produce bispecific antibodies. It's based on the somatic fusion of two different hybridoma cell lines expressing murine IgGs of desired specificities. However, the real percentage of functional bispecific antibody by a quadroma cell line is unpredictable and a laborious process is required to isolate the bispecific antibody from the side products.

By using molecular cloning technology, bispecific IgG antibodies can be assembled from two different heavy and light chains expressed in the same producer cell. The production of bispecific antibodies requires at least two plasmids for heterodimerized heavy chains and one plasmid for a common light chain or two light-chain plasmids if two different light chains are used. Notably, expressing HC and LC on separate plasmids is recommended because the manipulation of the plasmid ratio is an easy and efficient approach to optimize protein assembly for desired products. Subsequently, a laborious and time-consuming process is typically needed to select the most desirable clonal cell lines from a heterogeneous stable transfectant pool for large-scale antibody production.

Compared to stable transfections, transient transfection can deliver results in a few days without integrating recombinant DNA into the host genome. Human embryonic kidney (HEK293) and HEK-based Expi 293 cells are human cells for transient expression, which has been used early in bsAb development.

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.

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