Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach

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.

Chemical and Enzymatic Methods for Post-Translational Protein–Protein Conjugation

“…Benzaldehyde and hydrazine functional groups were introduced using NHS ester conjugation at lysine, and the resulting proteins were mixed in a 1:1 ratio, generating a hydrazone-linked species. 87 This approach was used to generate T cell recruiting bispecific antibodies to cancer cells overexpressing EGFR. However, it should be noted that hydrazones are not completely stable under aqueous conditions and can undergo hydrolysis.…”

“…A further lysine-selective method designed to overcome stability issues arising from disulfide or maleimide linkages was recently described. Benzaldehyde and hydrazine functional groups were introduced using NHS ester conjugation at lysine, and the resulting proteins were mixed in a 1:1 ratio, generating a hydrazone-linked species . This approach was used to generate T cell recruiting bispecific antibodies to cancer cells overexpressing EGFR.…”

Macrophage-engaging peptidic bispecific antibodies (pBsAbs) for immunotherapy via a facile bioconjugation strategy

“…The use of biological approaches to generate bispecic antibodies was started two decades ago [20][21][22] such as the knobs-intoholes strategy, 23 strand-exchange engineered domain (SEED) CH 3 heterodimers approach, 24 orthogonal Fab interface, 25 and CorssMab design. 26 Recently, chemical approaches [27][28][29][30][31][32][33][34][35] to develop bispecic antibodies has recently emerged, which shows advantages in the bispecic antibody production. In 2010, Doppalapudi et al introduced a novel technology for creating bispecic antibodies, termed bispecic CovX-Bodies, which are generated by fusing pharmacophore peptide heterodimers to a scaffold antibody that effectively targets both vascular endothelial growth factor and angiopoietin-2.…”

Double hit — bispecific antibodies in cancer therapy

“…Bispecific antibodies are generated through such techniques as chemical conjugation or genetic recombination and exhibit specific structures, compositions, and functional, biochemical, and pharmacological properties [3,4]. As a result, they exert effector functions that go beyond natural functions of antibodies, with applications spanning diagnostics, imaging, prophylaxis, and therapy [4].…”