An engineered selenocysteine defines a unique class of antibody derivatives

Abstract

Selenocysteine is cotranslationally inserted into proteins by recoding the stop codon UGA from termination to selenocysteine insertion. The nucleophilic selenol group of selenocysteine endows this rare amino acid with unique chemical reactivity that allows regiospecific covalent conjugation in the presence of the other natural amino acids. Using a mammalian expression system, we generated an IgG1-derived Fc fragment with a C-terminal selenocysteine in yields comparable to conventional monoclonal antibodies and conjugated it to an electrophilic derivative of a peptidomimetic that binds with high affinity and specificity to integrin ␣4␤1. Through this conjugation, both the biological and chemical components are endowed with pharmacological advantages. We demonstrate that whereas the Fc protein increases the circulatory half-life from minutes to days and mediates transcytosis through binding to the neonatal Fc receptor, the peptidomimetic introduces cross-species binding to cell surface integrin ␣4␤1 and blocks its interaction with vascular cell adhesion molecule-1. Compared with conventional monoclonal antibodies, our technology benefits economically from combining a generic biological component with a variable chemical component.antibody engineering ͉ Fc fragment ͉ neonatal Fc receptor ͉ small synthetic molecules ͉ integrin ␣4␤1

Selective chemical protein modification

“…Interestingly, use of bromomaleimides has opened up the possibility of reversible conjugation, allowing modulation of activity and in vivo monitoring, while also allowing the bridging and stabilisation of native disulfides 19,24 . The rare 21st amino acid, selenocysteine, can also be engineered into proteins and used to react with maleimides; greater Se nucleophilicity can allow conjugation selectivity over cysteine residues 25 .…”

Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry

“…Insertion of selenocysteine residues into proteins does not require synthetic tRNA, but only that the 3′ end of cDNA is modified to include a selenocysteine insertion sequence (24). Once a Sec-containing antibody is isolated, maleimide or iodoacetamide can be used to create selenoether conjugates (25).…”

Chemically Programmed Bispecific Antibodies That Recruit and Activate T Cells

“…Selective Conjugations-Established conditions for selective conjugation at the Sec interface were used (16,20). In brief, v9 Fab-Sec, v9 IgG-Sec and the previously described (16) Fc-Sec were diluted in 15 ml of 100 mM sodium acetate (pH 5.2) and concentrated to 4 M using a 10-kDa cutoff centrifugal filter device (Millipore).…”

“…This is due, in part, to improved methods for rational design and the ability to generate and screen large small molecule libraries (13,14). To equip small molecules with the pharmacokinetic and pharmacodynamic properties of mAbs, in particular, extended circulatory half-life and effector functions, chemical programming strategies have been developed that allow molecularly defined covalent docking of monospecific (15,16) or bispecific (17,18) small molecules that recognize cell surface receptors to antibody molecules with unique chemical reactivity. In addition to blending favorable features of small molecules and mAbs, chemically programmed mAbs are economically attractive because they utilize the same antibody construct for a virtually unlimited number of specificities, reducing production costs and shortening preclinical-to-clinical translation times (19).…”

“…Thus, the resulting conjugates are bispecific as they combine two different specificities in one molecule; one for CD3 mediated by the biological component and one for a cell surface receptor mediated by the chemical component. This work builds on our previously introduced chemical programming strategy that employs a Sec interface for the generation of molecularly defined antibody-small molecule conjugates in various formats (16,20). To demonstrate the feasibility and potency of chemically programmed biAbs, we first synthesized derivatives of small molecules (supplemental Fig.…”