Overview of Heavy Chain and Light-Chain Assembly

Heavy chain and light chain assembly is a method of generating bispecific antibodies based on genetic engineering techniques. Its core idea is to utilize the natural interaction between antibody heavy and light chains by introducing specific mutations into the heavy and light chains so that two different heavy and light chains can be correctly assembled into a bispecific antibody without producing erroneous combinations or hybrids. The advantage of this method is that it can maintain the natural structure and function of the antibodies, while avoiding the instability, heterogeneity and immunogenicity caused by chemical coupling or hybridoma methods. In addition, this method can also regulate the affinity and valency of the bispecific antibodies by changing the interaction strength between the heavy and light chains or between the heavy chains, thereby improving their biological activity and clinical efficacy.

The Principles of Heavy Chain and Light Chain Assembly

The principle of heavy chain and light chain assembly is to utilize the natural interaction between antibody heavy and light chains, by introducing specific mutations into the heavy and light chains, so that two different heavy and light chains can be correctly assembled into a bispecific antibody, without producing erroneous combinations or hybrids. The interaction between antibody heavy and light chains is mainly determined by two regions: one is the non-covalent bond between the heavy chain constant region (CH1) and the light chain constant region (CL), and the other is the hydrogen bond and hydrophobic interaction between the heavy chain variable region (VH) and the light chain variable region (VL). By introducing specific mutations into these regions, the interaction strength between the heavy and light chains can be changed, thereby achieving the orthogonal assembly of two different heavy and light chains.

Steps for Heavy Chain and Light Chain Assembly

The basic steps of heavy chain and light chain assembly are as follows:

• The first step is to select two monoclonal antibodies for the target antigens and clone their heavy and light chain genes into expression vectors. The vectors should contain elements for efficient expression and secretion of the chains.
• The second step is to introduce specific mutations into the heavy and light chain genes so that they can be orthogonally assembled into a bispecific antibody. The mutations can be introduced by various methods, such as mutagenesis and gene editing. The mutations can be selected based on literature or computer prediction.
• The third step is to transfect the vectors into mammalian cells and collect the bispecific antibodies from the culture medium. Mammalian cells can provide correct post-translational modifications and folding for the antibodies. Common mammalian cells include CHO, HEK293, NS0, etc.
• The fourth step is to purify, characterize, and functionally validate the bispecific antibodies. Purification aims to remove impurities and misassembled antibodies. Common methods include chromatography. Characterization aims to determine the structure, composition, purity, stability, etc., of the antibodies. Common methods include SDS-PAGE, mass spectrometry, etc. Functional validation aims to evaluate the affinity, valency, biological activity, etc., of the antibodies for the target antigens. Common methods include ELISA, flow cytometry, etc.

The Characteristic of Heavy Chain and Light Chain Assembly

The characteristic of heavy chain and light chain assembly is that it can maintain the natural structure and function of the antibodies while avoiding the instability, heterogeneity and immunogenicity caused by chemical coupling or hybridoma methods. In addition, this method can also regulate the affinity and valency of the bispecific antibodies by changing the interaction strength between the heavy and light chains or between the heavy chains, thereby improving their biological activity and clinical efficacy.

Table 1: Comparison of different methods for generating bispecific antibodies

As can be seen from Table 1, the heavy chain and light chain assembly has obvious advantages in purity, stability, safety and immunogenicity, while it is comparable to other genetic engineering methods in efficiency and cost. Therefore, heavy chain and light chain assembly is a suitable method for producing high-quality bispecific antibodies, especially for those applications that require maintaining the natural structure and function of the antibodies.

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