Endogenous coupling of amino acids and Disulfide re bridging strategy
Endogenous coupling of amino acids
One of the most common coupling methods is to use the lysine residue of the antibody, the amino acid nucleophilic NH2 group, to react with the electrophilic N-hydroxysuccinimide (NHS) Group on the lik payload. Although the reaction is simple, the high abundance of available lysine residues leads to the formation of uneven mixtures of many ADCs under random distribution. DAR is controlled by antibody-drug conjugates stoichiometry, which is widely used, including approved ADCs such as Besponsa, Mylotarg, and Kadcyla.
Disulfide re bridging strategy
IgG antibodies contain four disulfide bonds between chains, two connecting light chains and heavy chains, and two are located in the hinge region connecting two heavy chains. They maintain the integrity of monoclonal antibodies. Another classic biological coupling pathway explores the role of these cysteines as payload attachment points. The reduction of four disulfide bonds usually produces eight sulfhydryl groups, which can react with the linker of maleimide to produce ADC with DAR=8.
Dorona and colleagues reported an example of an ADC with a chimeric anti-CD30 monoclonal antibody coupled to MMAE, DAR= 8. Compared with the classical lysine coupling, this payload loading method is better controlled. However, it is reported that the plasma clearance rate will be higher and the risk of plasma aggregation will be reduced.
In 2015, chudasama et al. Introduced a new type of re bridging reagent, dibromopyridazinediones. They proved that it can be effectively inserted into the disulfide bond, and the resulting structure shows excellent hydrolytic stability even at high temperature. However, with the increase of temperature on the reduction step, heterogeneity is also observed, and this structure also allows the selective introduction of different functional groups.
Divinylpyrimidine is another effective re-bridging reagent, which can produce stable ADC with Dar = 4. Spring et al. Studied the effect of vinyl heteroaryl scaffold on cysteine re bridging. They believe that replacing pyridine with pyrimidine can make heteroaryl ring a better electron acceptor, so as to improve the crosslinking efficiency. Their work extends to divinyltriazine, which shows higher efficiency at high temperature.
In order to avoid the disadvantage of in vivo instability associated with classical maleimide coupling, Barbas et al studied methylsulfonylphenyloxadiazole, which has a specific reaction to cysteine. They are more stable than cysteine maleimide conjugates in plasma. Inspired by this, Zeglis designed dipods reagent, which contains two oxadiazole methyl sulfone parts connected by phenyl. Dipods forms covalent bonds with two sulfate radicals in the way of re bridging. Compared with maleimide coupling, coupling in this way has superior stability in vitro and performance in vivo.
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