Rapid and Efficient Generation of Stable Antibody–Drug Conjugates via an Encoded Cyclopropene and an Inverse‐Electron‐Demand Diels–Alder Reaction

Abstract

Homogeneous antibody–drug conjugates (ADCs), generated by site‐specific toxin linkage, show improved therapeutic indices with respect to traditional ADCs. However, current methods to produce site‐specific conjugates suffer from low protein expression, slow reaction kinetics, and low yields, or are limited to particular conjugation sites. Here we describe high yielding expression systems that efficiently incorporate a cyclopropene derivative of lysine (CypK) into antibodies through genetic‐code expansion. We express trastuzumab bearing CypK and conjugate tetrazine derivatives to the antibody. We show that the dihydropyridazine linkage resulting from the conjugation reaction is stable in serum, and generate an ADC bearing monomethyl auristatin E that selectively kills cells expressing a high level of HER2. Our results demonstrate that CypK is a minimal bioorthogonal handle for the rapid production of stable therapeutic protein conjugates.

Chemical Methods for Selective Labeling of Proteins

“…In parallel, Lin's group reported the insertion of a cyclopropene derivative to react with a 1,3‐dipole generated by photoactivation of tetrazoles . Also, Chin and co‐workers synthesized cyclopropene engineered trastuzumab and used it for chemoselective conjugation with tetrazine derivative of MMAE drug . The capability of tetrazine to enable inverse‐electron‐demand Diels‐Alder reaction was also harnessed by Guo and co‐workers through a pre‐engineered styrene (ncAA) .…”

“…[36] Also, Chin and co-workers synthesized cyclopropene engineered trastuzumab and used it for chemoselective conjugation with tetrazine derivative of MMAE drug. [37] The capability of tetrazine to enable inverse-electron-demand Diels-Alder reaction was also harnessed by Guo and co-workers through a pre-engineered styrene (ncAA). [38] In an interesting report, Pentelute's group synthesized proteins with an electrophilic selenium group primed for late-stage installation of probes (Scheme 4e).…”

Bioorthogonal Ligation and Cleavage by Reactions of Chloroquinoxalines with ortho‐Dithiophenols

“…Bioorthogonal chemistry has become ap owerful tool in chemical biology [1] (the concept of bioorthogonal chemistry was first proposed by Bertozzi in 2003 [2] ), and it shows wide applications such as chemical labelling of biomolecules in living cells, [3] post-translational modification of proteins, [4] and construction of antibody drug conjugates. [5] Therefore,t he development of highly efficient bioorthogonal reactions is of great importance.T he bioorthogonal reactions should meet conditions including fast rate,high yield, good solubility,and high stability of the reactants and product(s) under physiological conditions,n on-toxicity to the biological system, minimizing steric interactions with the biomolecule,a nd facilitating incorporation by the endogenous cellular machinery. [6] An umber of bioorthogonal reactions including bioorthogonal ligations and cleavages have been developed thus far.T he most representative bioorthogonal ligations include the native chemical ligation of proteins, [7] protein labeling through the reaction of bisarsenical dyes with ag enetically incorporated tetracysteine unit, [8] theb iocompatible Staudinger ligation reaction of azides with modified triphenylphosphines, [9] Cu I -catalyzed azide-alkyne cycloaddi-tion, [10] strain-promoted azide-alkyne cycloaddition, [11] the inverse electron-demand Diels-Alder (IED-DA) reaction, [12] and boronate formation.…”

“…

Ab ioorthogonal ligation and cleavage method via reactions of chloroquinoxalines (CQ) and ortho-dithiophenols (DT) is presented. Double nucleophilic substitutions of orthodithiophenols to chloroquinoxalines provide conjugates containing tetracyclic benzo [5,6][1,4]dithiino[2,3-b]quinoxaline with strong built-in fluorescence together with release of the other functional molecules.T hree cleavable linkers were designed and successfully used in release of the molecules containing biotin from the protein conjugates.T he CQ-DT bioorthogonal reactions can be applied for the bioorthogonal ligations,b ioorthogonal cleavages,a nd trans-tagging of proteins,a nd show advantages of readily accessible unnatural orthogonal groups,a ppealing reaction kinetics (k 2 % 1.3 m À1 s À1 ), excellent biocompatibility of orthogonal groups, and high stability of conjugates.T his complements previous bioorthogonal reactions and is anew route for protein-fishing applications and in-gel fluorescence analysis.

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Efficient Phage Display with Multiple Distinct Non‐Canonical Amino Acids Using Orthogonal Ribosome‐Mediated Genetic Code Expansion

“…We initially introduced an amber codon at position 252 in the scFv (118 EU numbering), creating an ScFv(252TAG)-g3 gene;this site is commonly used to modify antibodies without affecting antigen binding. [15] Production of phage from cells bearing phagemid-based systems is dependent on helper phage infection to provide phage coat proteins. [1b, 2b] All [10] but one [4b] report of using MjTy rRS/tRNA CUA derivatives to incorporate ncAAs rely on ah elper phage in which g3 has been deleted (dM13);t his makes the production of phage particles dependent on expression of the full length p3 fusion from the phagemid vector and phage titers are ncAA dependent in these systems.…”