Synthesis of bispecific antibodies using genetically encoded unnatural amino acids

Bispecific antibodies were constructed using genetically encoded unnatural amino acids with orthogonal chemical reactivity. A two-step process afforded homogeneous products in excellent yield. Using this approach, we synthesized an anti-HER2/anti-CD3 bispecific antibody, which efficiently cross-linked HER2+ cells and CD3+ cells. In vitro effector-cell mediated cytotoxicity was observed at picomolar concentrations.     

Selective formation of covalent protein heterodimers with an unnatural amino acid

We report a strategy for the generation of heterodimeric protein conjugates using an unnatural amino acid with orthogonal reactivity. This paper addresses the challenges of site-specificity and homogeneity with respect to the synthesis of bivalent proteins and antibody-drug conjugates. There are numerous antibody-drug conjugates in preclinical and clinical development, yet these are based either on nonspecific lysine coupling chemistry or on disulfide modification made difficult by the large number of cysteines in antibodies. Here, we describe a recombinant approach that can be used to rapidly generate a variety of constructs with defined conjugation sites. Moreover, this methodology results in homogeneous antibody conjugates whose biological, physical, and pharmacological properties can be quantitatively assessed and subsequently optimized. As proof of concept, we have generated anti-Her2 Fab-Saporin conjugates that demonstrate excellent potency in vitro.