ABSTRACT
Antibody-drug conjugates (ADCs) are fulfilling the promise of targeted therapy with meaningful clinical success. An intense research effort is directed towards improving pharmacokinetic profiles, toxicity and chemical stability of ADCs. The majority of ADCs use amide and thioether chemistry to link potent cytotoxic agents to antibodies via endogenous lysine and cysteine residues. While maleimide-cysteine conjugation is used for many clinical stage ADC programs, maleimides have been shown to exhibit some degree of post-conjugation instability. Previous research with site-directed mutagenic incorporation of cysteine residues for conjugation revealed that the stability of the drug-antibody linkage depends on the site of conjugation. Here we report on a collection of engineered cysteine antibodies (S239C, E269C, K326C and A327C) that can be site-specifically conjugated to potent cytotoxic agents to produce homogenous 2-loaded ADCs. These ADCs confirm that site of conjugation impacts maleimide stability and present a novel mechanism of thioether stabilization, effectively unlinking stability from either local chemical environment or calculated solvent accessibility and expanding the current paradigm for ADC drug-linker stability. These ADCs show potent in vitro and in vivo activity while delivering half of the molar equivalent dose of drug per antibody when compared to an average 4-loaded ADC. In addition, our lead engineered site shields highly hydrophobic drugs, enabling conjugation, formulation and clinical use of otherwise intractable chemotypes.
Subject(s)
Cytotoxins , Protein Engineering/methods , Single-Chain Antibodies , Animals , Cytotoxins/biosynthesis , Cytotoxins/chemistry , Cytotoxins/isolation & purification , Cytotoxins/pharmacology , Female , Humans , Immunoconjugates/chemistry , Immunoconjugates/isolation & purification , Immunoconjugates/pharmacology , Mice , Mice, Nude , Rats , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/pharmacologySubject(s)
Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Prostaglandins G/chemistry , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Catalysis , Cyclooxygenase 2 , Electron Spin Resonance Spectroscopy , Isoenzymes/chemistry , Molecular Conformation , Prostaglandin H2 , Prostaglandin-Endoperoxide Synthases/chemistry , Prostaglandins G/chemical synthesis , Prostaglandins H/biosynthesis , Prostaglandins H/chemistry , Structure-Activity RelationshipABSTRACT
Useful methodology is described for the synthesis of dehydroalanine residues (II) within peptides. The unnatural amino acid (Se)-phenylselenocysteine (I) can be incorporated into growing peptide chains via standard peptide synthesis procedures. Subsequent oxidative elimination affords a dehydroalanine at the desired position. The oxidation conditions are mild and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. To illustrate its utility, cyclic lanthionines have been synthesized by this method.
Subject(s)
Alanine/analogs & derivatives , Peptides/chemical synthesis , Alanine/chemistry , Oxidation-Reduction , Peptides/chemistry , Selenocysteine/analogs & derivatives , Selenocysteine/chemistry , StereoisomerismABSTRACT
Recent crystallographic and biochemical studies have revealed the existence of numerous novel post-translational modifications within enzyme active sites. These modifications create structural and functional diversity. Although the function and biosynthesis of some of these modifications are well understood, others need further investigation.
