Abstract
Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues—which can be chemical conjugation sites—were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.
Highlights
Antibodies are composed of several structurally and functionally independent domains
For the site-specific chemical conjugation, a single mutated residue in the framework region (FR) of the 0K single chain Fv (scFv) was returned to lysine: the 19th residue in the VH domain and the 103rd residue in the VL domain was independently returned to lysine
We showed the potential of using lysine-free block modules for homogeneously assembling different modules to generate chimeric proteins that cannot be formed by means of genetic fusion approaches
Summary
Antibodies are composed of several structurally and functionally independent domains They can be fragmented and their block modules can be rearranged to form recombinant proteins with non-native structures and functions [1]. Bispecific antibodies with two different Fvs have been constructed from two distinct monoclonal antibodies, and they have been used to induce interactions between two types of cell by forming linkages between the two target antigens on the surfaces of the cells [11,12,13,14]. Advances in recombinant genetic fusion approaches have enabled the production of homogenous bispecific antibody molecules, and various bispecific antibodies have been genetically designed with antibody fragments as block modules [18]. Crosslinking techniques, which provide a wider variety of steric arrangements of block modules, would enable us to find appropriate linkages between the block modules of Fvs to generate functional bispecific antibodies
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