An ADC consists of an antibody and toxin bridged by a linker that affects the coupling stability to some extent and has an important influence on ADC efficacy.
The premature release of toxins from ADC have two possible effects:
- The toxin that has fallen off before reaching the target position will damage healthy cells.
- The loss of toxins makes ADC drugs lose their efficacy.
Finding a suitable linker is an important factor in the successful development of ADC. The first ADC drug on the market chose a relatively inert connector—Hydrazone to avoid unstable connections. Even if Hydrazone is selected, Mylotarg still kept hydrolyzing and losing payloads.
In order to avoid the instability caused by the above linkers, researchers subsequently developed the most widely used linker—Maleimide that produces a relatively stable thioether bond through the covalent binding of Michael receptor and cysteine sulfhydryl group on the antibody.
However, the stability of thioether bond is relative. When encountering other amino acid residues containing mercaptan with similar nucleophilicity, the linker exchange reaction will also occur, resulting in the loss of ADC payloads. The Maleimide linker first underwent reverse Michael addition, and then exogenous mercaptan (cysteine and GSH on serum albumin) was added to Maleimide, resulting in about 75% payload transfer loss of the research substrate.
More linkers need to be developed to solve such problems.
Ⅰ. Hydrolyzed Maleimide linker
When the payload transfer occurs in the Maleimide joint, it will also be hydrolyzed very slowly, and it is difficult for the hydrolyzed product to undergo reverse Michael addition, which may be due to the weakening of proton activation of carbonyl α position after Maleimide hydrolysis that is not easy to eliminate.
If an ADC’s total antibody clearance rate in the blood is slow and the linker and antibody joint stability is improved, the biological activity of the ADC will be enhanced.
Therefore, hydrolyzed Maleimide may be an ideal choice, which retains the advantages of mature Maleimide while avoiding its disadvantages.
The synthesized ADC with open ring can not conjugate with antibody using openring linker directly because hydrolyzed Maleimide has increased stability, which means a decreased reaction activity, and the hydrolytic ring opening could only be carried out after antibody conjugation.
It was found that the hydrolysis rate of the substrate MC structure was very slow, while the hydrolysis rate increased significantly after embedding 6 PEG units in the MC substrate.
It is speculated that the reaction mechanism is that the oxygen on the first PEG at the Maleimide joint will coordinate with the water molecule, guiding the water molecule to attack the carbonyl group from the optimal point, thus accelerating the hydrolysis.
In the experiment, trastuzumab targeting Her2 was selected to dismantle the disulfide bond with TCEP and couple with LP (linker+payload) to obtain 1-6 structure, which then hydrolyzed in different buffer systems, and the process of light chain modification with small molecular weight was monitored by MS.
The molecular weight of light chain + LP is 24540, and the molecular weight of hydrolysis increases gradually as hydrolysis proceeded, and only 30% is transformed after PH=7.4 reaction for 16h, while in PH=9.2 reaction for 14h, the conversion is basically complete.
The conversion of ADC without PEG unit was only 54% after PH=9.2 reaction for 14h.
The binding affinity of hydrolyzed and unhydrolyzed ADC to Her2 antigen is roughly the same as that of unmodified trastuzumab, but the hydrolyzed ADC has higher in vitro stability, better selectivity, better PK exposure, and better efficacy.
Ⅱ.Weaker new structure of Michael receptor
In order to avoid the inverse Michael reaction, we can try to choose the more inert Michael receptor substrate as the joint. The structural activity of alkyne and amide is weak, which seem to be a suitable choice.
Modified cysteine 1, cysteine 2 and glutathione 3 were used as substrates and connectors of nucleophiles for conjugation reaction. When PH=7.4, the reaction completed in 30~70min.
Because the addition of alkyne and mercaptan is trans-addition, the Z configuration is dominant, and the mercaptan and joint adduct are more stable than Maleimide joint in the presence of acid-base and excess mercaptan.