Bispecific antibodies (bsAbs) are antibodies that can bind two different antigens or epitopes simultaneously, thereby expanding the specificity and functionality of conventional monoclonal antibodies (mAbs). BsAbs have several advantages over mAbs, such as enhanced cytotoxicity, reduced resistance, novel mechanisms of action, and potential applications in cancer immunotherapy and other diseases. The first bsAbs were generated by chemical methods, such as cross-linking or reduction of mAbs, in the 1960s and 1970s. However, these methods produced bsAbs with low purity, high heterogeneity, and poor stability, limiting their clinical utility. Since then, various techniques and methods have been developed to generate bsAbs by genetic engineering, such as quadroma (or hybrid-hybridoma) technology, heavy-chain assembly, heavy-chain and light-chain assembly, and co-culture method. These methods aim to produce bsAbs with native IgG structure and function, which consist of two heavy-chains and two light-chains, and have an Fc region that can mediate effector functions and extend the serum half-life. However, generating bsAbs by Fc heterodimerization is not trivial, as it involves overcoming the natural tendency of the heavy-chains to form homodimers rather than heterodimers. Therefore, different strategies have been devised to engineer the Fc region of the heavy-chains to promote heterodimerization and prevent homodimerization. Moreover, the light-chains also need to be engineered to pair correctly with the corresponding heavy-chains and to avoid unwanted interactions or mispairing.
Quadroma technology is one of the earliest and simplest methods to generate bsAbs by Fc heterodimerization. Quadroma technology relies on the random association of the heavy-chains and light-chains from the two parental hybridomas, resulting in a mixture of antibodies with different specificities and valencies. Among these antibodies, only a small fraction (about 5–10%) are bsAbs with the desired specificity and functionality. The main advantages of quadroma technology are its simplicity, low cost, and high yield, as it does not require any genetic manipulation or sophisticated equipment. Moreover, quadroma technology can produce bsAbs with native IgG structure and function, which have an Fc region that can mediate effector functions and extend the serum half-life. The main disadvantages of quadroma technology are its low purity, high heterogeneity, and difficulty in screening and purification. As quadroma technology produces a complex mixture of antibodies, it is challenging to isolate and identify the bsAbs with the desired specificity and functionality. Moreover, quadroma technology is limited by the availability and compatibility of the parental hybridomas and the stability and productivity of the quadromas. Quadroma technology is still widely used for the generation of bsAbs, but it faces increasing competition from other methods that can produce bsAbs with higher purity, homogeneity, and stability. Some possible ways to improve quadroma technology are to use gene editing, cell sorting, or affinity chromatography to enhance the selection and purification of bsAbs, or to use alternative cell lines or hosts to increase the compatibility and productivity of quadromas.
Heavy-chain assembly is one of the most advanced and popular methods to generate bsAbs by Fc heterodimerization. Heavy-chain assembly relies on the introduction of mutations, deletions, or insertions in the CH3 domain of the Fc region, which is responsible for the formation of the heavy-chain dimer. These modifications create complementary interfaces or interactions between the heavy-chains, such as knobs-into-holes (KIH), electrostatic steering, CrossMab, DuoBody, and BiXAb, and prevent the formation of homodimers by steric hindrance or charge repulsion. The main advantages of heavy-chain assembly are its high purity, homogeneity, and stability, as it produces bsAbs with defined and consistent structure and function. Moreover, heavy-chain assembly can produce bsAbs with native IgG structure and function, which have an Fc region that can mediate effector functions and extend the serum half-life. Furthermore, heavy-chain assembly can be applied to various formats and platforms of bsAbs and can be compatible with different expression systems. The main disadvantages of heavy-chain assembly are its complexity, cost, and immunogenicity. As heavy-chain assembly involves genetic engineering and sophisticated equipment, it is more complex and costly than other methods. Moreover, heavy-chain assembly may introduce novel epitopes or alter the glycosylation pattern of the Fc region, which may increase the immunogenicity or reduce the efficacy of bsAbs.
Heavy-chain and light-chain assembly is another advanced and popular method to generate bsAbs by Fc heterodimerization. heavy-chain and light-chain assembly relies on the introduction of mutations, deletions, or insertions in both the CH3 domain of the Fc region and the CL domain of the light-chains, which are responsible for the formation of the heavy-chain dimer and the light-chain pairing, respectively. These modifications create complementary interfaces or interactions between the heavy-chains and the light-chains, such as common light-chain, dual variable domain, and single-chain variable fragment (scFv), and prevent the formation of homodimers or mispairing by steric hindrance or charge repulsion. The main advantages of heavy-chain and light-chain assembly are its high purity, homogeneity, and stability, as it produces bsAbs with defined and consistent structure and function. Moreover, heavy-chain and light-chain assembly can produce bsAbs with native IgG structure and function, which have an Fc region that can mediate effector functions and extend the serum half-life. The main disadvantages of heavy-chain and light-chain assembly are its complexity, cost, and immunogenicity. As heavy-chain and light-chain assembly involves genetic engineering and sophisticated equipment, it is more complex and costly than other methods. Moreover, heavy-chain and light-chain assembly may introduce novel epitopes or alter the glycosylation pattern of the Fc region or the light-chains, which may increase the immunogenicity or reduce the efficacy of bsAbs.
The co-culture method is another simple and flexible method to generate bsAbs by Fc heterodimerization. It relies on the co-expression of two different mAbs in separate cells, such as CHO cells, B cells, or hybridomas, and then co-culturing them in the same medium, where the antibodies can associate randomly to form bsAbs. The ratio of the two cell populations can be adjusted to control the yield and specificity of bsAbs. The main advantages of co-culture method are its simplicity, flexibility, and high yield, as it does not require any genetic manipulation or sophisticated equipment. Moreover, the co-culture method can produce bsAbs with native IgG structure and function, which have an Fc region that can mediate effector functions and extend the serum half-life. The main disadvantages of the co-culture method are its low purity, high heterogeneity, and difficulty in screening and purification. As the co-culture method produces a complex mixture of antibodies, it is challenging to isolate and identify the bsAbs with the desired specificity and functionality. Moreover, co-culture method is limited by the availability and compatibility of the parental cells and the stability and productivity of the co-culture. Some possible ways to improve the co-culture method are to use gene editing, cell sorting, or affinity chromatography to enhance the selection and purification of bsAbs, or to use alternative cell lines or hosts to increase the compatibility and productivity of co-culture.
References
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