Antibody-drug conjugates (ADCs) have become a promising targeted therapy strategy that combines the specificity, favorable pharmacokinetics and biodistributions of antibodies with the destructive potential of highly potent drugs. for conjugating (Scheme 1). After reduction of the disulfide bonds, the mutated monoclonal antibodies with the reduced number of interchain cysteines were conjugated with the drug vcMMAE. Through this method, homogenous antibody-drug conjugates with clear attachment sites could be produced. Scheme 1 Interchain cysteine to serine mutagenesis enables drugs to conjugate to the remaining cysteines. Adapted from reference [18]. Reducing the disulfide bonds of a monoclonal antibody should not affect its functions [19]. What SCH-527123 is more, interchain disulfide bonds are easier to be reduced than intrachain disulfide bonds [20]. These allow free thiol groups to be generated under mild reducing conditions while leaving the antibody intact at the same time. Liu [21] took advantage of the fact that different disulfide bonds in a monoclonal antibody have different susceptibilities towards reduction and developed another strategy to tightly control the site of conjugation. Limited reduction with TCEP or DTT predominantly yielded conjugates in which drugs were attached to heavy-light chain disulfides; partial re-oxidation of fully reduced antibodies with SCH-527123 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) yielded conjugates that drugs were mainly attached to by heavy-heavy chain disulfides [13]. 2.1.1. Addition to MaleimidesClassically, cysteine residues can be modified through addition of thiols to electrophiles such as maleimides (Scheme 2) [22,23,24,25]. The conjugate could be achieved by reducing the disulfide bonds of the antibody and then adding to maleimides. Addition to maleimides is the most common method for attaching drugs to antibodies. Adcetris?, which was approved by the FDA for the treatment of patients with Hodgkins lymphoma after failed autologous stem cell transplantation or patients with systemic anaplastic large-cell lymphoma after the failure of at least one prior multi-agent chemotherapy regimen, was produced by this method in which a maleimide-functionalized drug was conjugated to the interchain cysteine residues of an anti-CD30 antibody [15]. Maleimide-based antibody-drug conjugates were recently found to have limited stability in blood circulation [26], which would lower the efficacy of the conjugates and damage healthy tissue. Succinimide or maleimide hydrolysis is a promising method to get around this problem. Once hydrolyzed, the antibody-drug conjugates were no longer subject to elimination reactions of maleimides through retro-Michael reactions, thus improving the stabilities and potencies of ADCs [27,28,29]. Scheme 2 The synthesis of antibody-drug conjugates (ADCs) through the addition of thiols to maleimides. Adapted from reference [23]. 2.1.2. Disulfide-Thiol ExchangeThe approach disulfide-thiol exchange could also be used to synthesis ADCs by forming a new disulfide bond between drugs and antibodies [30,31]. Ojima [30] designed and synthesized novel antibody-taxoid conjugates that include highly cytotoxic taxoid drug and monoclonal antibodies that could recognize the EGFR expressed in cancer cells. In this study, taxoid bearing a free thiol group was attached to the pyridyldithio groups of the modified anti-EGFR antibodies through disulfide-thiol exchange (Scheme 3). The resulting conjugates possess remarkable antitumor activities against EGFR-expressing A431 (human epidermoid) tumor xenografts in immune deficient mice. Scheme 3 Preparation of antibody-taxoid conjugates via disulfide-thiol exchange. Adapted from reference [30]. 2.1.3. Addition to AlkynesTo avoid the maleimide instability issue, Kolodych [32] developed a heterobifunctional reagent, sodium 4-((4-(cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), for amine-to-thiol coupling (Scheme 4). This FLNC SCH-527123 reagent comprises a 3-arylpropionitrile (APN) group that replaces the maleimide and allows for the preparation of remarkably stable conjugates. Addition of thiols in the antibodies to the 3-arylpropionitriles predominantly produced [37] reduced all the disulfide bonds, exposing eight cysteine residues, then similarly used dibromomaleimide (DBM) to react with the free thiol groups of the antibody and produced a dithiomaleimide (DTM) ADC. Four cytotoxic drugs with this functional linker were attached to the monoclonal antibodies conveniently by linking with the cysteine residues. Chudasama and coworkers [27,38,39,40] presented a significant method towards next-generation antibody-based therapeutics through disulfide re-bridging. In their works, the reduction of disulfides and disulfide re-bridging could be achieved in one step by the use of a single reagent: dithioaryl(TCEP)pyridazinedione [38]. Disulfide re-bridging through the use of dibromopyridazinedione derivatives after disulfide reduction by.